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Advances in Tendon and Ligament Tissue Engineering: Materials Perspective.

1. Introduction

Traumatic tendon and ligamentous injuries represent significant healthcare and economic challenges for the future. Notably, these injuries are estimated to affect 110 million people in the United States [1], and incomplete repair is associated with variable disabilities and chronic sequelae [2, 3].

Tendon represents a specialised connective tissue in which collagen type I accounts for ~80% of the net dry weight. In combination with proteoglycans and elastin, collagen permits high mechanical strength in tendons [4, 5]. Furthermore, tendons display a unique structural hierarchy where collagen molecules produce collagen fibrils, which group together to form collagen fibres. The multicomposite tendon units are composed of several collagen fibres, known as tropocollagen (Figure 1) [3, 4, 6].

Ligaments are another form of viscoelastic connective tissue, with a highly organised composition where collagens (types I, III, and V) constitute the bulk. Proteoglycans and chondroitin sulfate are also expressed, allowing the ligament tissue to swell in aqueous environments [7]. In the body, the attachment of tendons and ligaments to bone involves a transition zone with unmineralised and mineralised fibrocartilage [3, 8]. Tendons can further attach to muscles through fascia [4]. Defining the structural organisation of tendons and ligaments has improved understanding of the way these heterogeneous tissues function in synergy [9].

Surgical repair of tendons and ligaments via primary suture or autologous transfer techniques are considered the gold standard modality of care. However, these solutions are often associated with challenges including reduced mechanical strength due to scar tissue formation, infections, donor site morbidity, and limited availability of autografts [10]. Furthermore, specialised physiotherapy protocols are required to improve the repair-site properties and limit scar tissue formation [11].

To overcome this clinical issue, additional therapeutic options involving the use of synthetic prosthesis or tissue engineered biomaterial constructs have been investigated in the literature [12]. Ranges of synthetic prosthetic devices have been described as tendon and ligament tissue substitutes. Nonabsorbable and biocompatible polyester polyethylene terephthalate (PET) was investigated as a potential tendon and ligament tissue prosthesis material with suitable mechanical and tissue integrative properties [13, 14]. Other materials like polytetrafluoroethylene (PTFE) were also investigated as ligament tissue substitutes or in tendon augmentation grafts, for their biologically inert and strong mechanical characteristics [15, 16]. However, limitations such as graft failure, poor durability, poor tissue integration, and foreign body synovitis were observed with these materials [17]. Tissue engineering represents an alternative option with potential for proper tissue integration of implants.

Different synthetic or naturally derived materials have been investigated as scaffolds, which permit cell integration and subsequent matrix deposition. Among the naturally derived materials, collagen and chitosan have been extensively investigated for scaffold development due to their optimum biocompatibility and tissue integration potential [18]. Nevertheless, these materials exhibit limitations such as low mechanical strength, batch variability, and difficult processing with latent immunogenicity [19].

Synthetic materials, including polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-co-glycolic acid (PLGA) have been extensively investigated for tendon and ligament repair [20]. Synthetics exhibit several advantages linked to large-scale manufacturing, limited disease transmission, controlled degradation, and better host tissue integration. There are also limitations associated with synthetic biomaterials; cell integration is challenging without further material treatment, degradation-related products can be cytotoxic, and the materials are mechanically weaker than healthy musculoskeletal tissues [18, 21].

Novel tissue engineering approaches using composite materials have also been described to mimic the complex, nonhomogenous environments in tendons and ligaments. In these composite materials, specialised cells have been combined with biomaterials to produce complex, heterogeneous scaffolds with controlled mechanical properties [1, 9]. This review will present an overview of the different approaches described to address the use of composite scaffolds for tendon and ligament tissue engineering. An in-depth critique of the mechanical properties, cell/ tissue integration, success criteria, and limitations is discussed in detail.

2. Materials and Methodology

Comprehensive literature searches were conducted on the PubMed, Medline, Web of Science, and Google Scholar databases. Various combinations of keywords were used in the search process, including "tendon", "ligament", "tissue", "engineering", "hybrid", "composite", "scaffold", "material", "biomaterial", "graft", and "polymer". Only publications in English were included and there were no restrictions on year of publication since no previous reviews were found covering the use of composite materials in tendon and ligament tissue engineering simultaneously. All relevant manuscripts were accessed and articles that combined tissue engineering approaches for both tendons and ligaments were included. Articles that discussed different tissue engineering approaches of the tendon/ligament to bone interface were also excluded due to the broadness of the topic and the important number of review articles on the subject [8, 22-24].

3. Results and Discussion

3.1. Composite Scaffolds for Tendon and Ligament Tissue Engineering. Tendon injuries present significant healthcare challenges due to slow healing rates, loss of function, and scar tissue formation around trauma sites [12]. In severe cases, tissue engineered scaffolds have been fabricated to replace the lost tissues. These artificial grafts can be composed of natural or synthetic biomaterials [49]. The ideal scaffold in tendon tissue engineering should exhibit specific properties such as controlled degradation rates, appropriate mechanical properties, nonimmunogenicity, and suturability. Additionally, easy mass processing and fabrication, while mimicking the native tissue environment, are desirable [12].

3.2. Synthetic Composite Materials. One example of materials used for tendon tissue engineering involved the fabrication of composite, heterogeneous scaffolds from polyglycolic acid (PGA), and polylactic acid (PLA) [44]. The outer surface was composed of a knitted scaffold made from a 4:2 mixture of PGA and PLA fibres. Internally, the construct contained longitudinally arranged, unwoven PGA fibres. The whole construct was folded and secured with sutures at each end to produce a cord structure and was tested both in vitro and in vivo. Results showed that the scaffold seeded with adipose-derived stem cells (ADSCs) promoted matrix deposition and the formation of mature collagen fibrils. These scaffolds presented subphysiological mechanical properties. Although the study showed promising results, assessment of tenogenic differentiation of ADSCs was not elaborated.

Another manufacturing technique was proposed by Baker B. M. et al., who investigated the use of coelectrospinning technique of different polymers to yield a composite scaffold with variable degradation techniques [28]. They evaluated the use of PCL fibres as slow absorbing elements, while PLGA (50:50 polylactic/ glycolic acid) or PCL/ PLGA fibres were applied for intermediate degradation rates in a single scaffold. A water-soluble polyethylene oxide (PEO) was as a sacrificial fibre element with fast absorption rates and aimed to increase the scaffold porosity with minimal influence on scaffold integrity. Mechanical assessment of PCL/ PLGA/ PEO constructs showed a maximum stress yield of nearly 3.5 MPa at 0.08% strain. The modulus was around 100 MPa and the yield strain was 0.026. PCL/ PLGAPCL/ PEO scaffolds showed a maximum stress yield of 2 MPa at 0.12% strain. Initial modulus of the material was 25 MPa and dropped to 12.5 MPa after 63-day incubation in culture media. Strain increased from 0.065% to 0.12% after incubation. A 22% mass decrease of the composite scaffold after hydration was caused by the dissolution of the PEO component. However, the mechanical properties change after hydration and dissolution of PEO are worth further explanation.

It has been suggested that woven scaffolds are superior for tissue integration due to their interconnected porous structures. Nevertheless, they require challenging cell seeding techniques and complex cell delivery systems [4, 5]. Sahoo S. et al. investigated the use of woven scaffolds made from PLGA or PLLA [29]. Both f scaffolds were coated with PCL, PLGA nanofiber, or collagen type I to yield composite scaffolds and were seeded with porcine bone marrow-derived MSCs. Some collagen-coated scaffolds were seeded with human dermal fibroblasts to test cell seeding and integration efficiency. Results showed that PLLA-based woven scaffolds performed inferiorly in terms of cell attachment. This was linked to the hydrophobic nature of the PLLA material. Furthermore, PCL coating of both types of knitted scaffolds was associated with higher mechanical strength but reduced cell attachment. This was also linked to greater hydrophobicity in PCL than in the other polymers.

As mechanical strength is particularly important, an approach using PLA with graphene nanoplatelets (GNP) and PLA with carboxyl functionalized carbon nanotubes (CNT-COOH) has been proposed by Pinto et al. [25, 26]. In vitro assessment of the composite was nontoxic to human fibroblasts. In vivo assessment using mouse model did not show any toxicity or local or systemic inflammatory response. The addition of nanofillers mentioned enhanced the mechanical properties of PLA polymer films reaching Young's modulus of 4.86 [+ or -] 0.47 GPa for CNT-COOH scaffolds and 4.92 [+ or -] 0.15 GPa for GNP scaffold groups. The tensile strength however was mostly enhanced in the PLA/ CNT-COOH scaffolds reaching up to 72.22 [+ or -] 1.52MPa. The authors did not investigate, however, the effect of in vivo implantation on the associated mechanical properties mimicking real clinical settings.

3.3. Biological Composite Scaffolds. Collagen type I composite scaffold was also investigated to incorporate resilin-like protein. Resilin is an arthropods protein with elastic and highly stretchable structure. Sanami, M., et al. [27] investigated the fabrication of such composite. The scaffold was made through extrusion process of composite solution containing Collagen and Resilin at different concentration into polyethylene glycol buffer. Fibres were subsequently cross-linked using 4-arm poly(ethylene glycol) ether tetrasuccinimidyl glutarate solution. Mechanical assessment showed that resilin in non-cross-linked collagen scaffold significantly reduced stress at break and Young's modulus values, while significantly increasing break strain. Cross-linked collagen/resilin scaffold showed significant increase in stress and strain values and a significantly decreased Young's modulus values. This shows an interesting effect of resilin exhibiting its natural properties. In vitro, the scaffold produced supported 100% fibroblast proliferation and alignment compared to 80% in collagenfibre control.

Scaffolds made from collagen and glycosaminoglycan (GAG) were recognised for their role in supporting cellular proliferation and differentiation. However, this type of scaffold lacks the mechanical characteristics required for tendon tissue engineering applications. Caliari et al., 2011, have proposed the concept of developing composite materials in a core-shell fashion with the necessary mechanical properties [30]. The group fabricated scaffolds composed of highly porous, aligned isotropic GAG cores surrounded by strong, high density isotropic GAG membranes. The core-shell design was proposed to increase the mechanical strength of the scaffolds. The material was fabricated using an evaporation technique to create membranes and freeze-drying to incorporate the core within the membrane shell. Dehydrothermal (DTH) cross-linking was used to increase material integration, as well as the mechanical properties. In vitro assessment using horse-derived tenocytes demonstrated good cell attachment, proliferation, and cell viability up to 14 days after seeding. Scaffolds exhibited high porosity and appropriate mechanical properties depending on the membrane thickness. Several factors however that need further investigation like cellular functional assessment (e.g., protein expression), nature and content of collagen after cell seeding, and the mechanical properties of the scaffold at different states (e.g., wet versus dry).

Chitosan is a naturally derived polysaccharide with excellent potential for tissue engineering applications. Due to the biocompatible and cell adhesive properties of chitosan, the polysaccharide has been investigated for tendon tissue regeneration [9-11]. In one example, composite scaffolds made from chitosan and alginates were produced through a spinning/coagulation technique to yield an alginate-0.1% chitosan scaffold. Alginate is an anionic polysaccharide with calcium chain in which the integration of chitosan improves its biocompatibility and cell adhesive potential and decreases its degradation rate. In vitro assessment using rabbit patellar tendon fibroblasts showed that alginate-0.1% chitosan scaffolds had significantly higher cell adhesion and matrix deposition compared to alginate-only and polyglactin 910 controls. The alginate-0.1% chitosan material was evaluated mechanically and exhibited lower tensile strength and strain at failure than the polyglactin group [33]. Chitosan was also fabricated with hyaluronic acid followed by wet spinning and hybridisation to increase the mechanical properties of the construct. Different hyaluronic acid concentrations were investigated and showed that the combination of chitosan with 0.1% hyaluronic acid showed the best cell attachment. The final composite constructs were sterilised using ethylene oxide gas prior to in vitro evaluation with rabbit patellar fibroblasts. Mechanical properties of the materials were reduced within the first 2 hours after seeding but the consequent modulus was maintained for 28 days of culture. Cell proliferation quantification using DNA content analysis showed significant improvement in chitosan-0.1% hyaluronic acid composites compared to other chitosan-based scaffolds. To determine the clinical potential of the composite scaffolds, authors assessed the chitosan-0.1% hyaluronic acid composites in vivo by treating rabbit rotator cuff injuries with cell-seeded scaffolds [31]. The scaffolds were cultured with rabbit patellar tendon fibroblasts for 4 weeks prior to implantation. Additionally, authors tested the potential for ligament tissue engineering using a rabbit medial collateral ligament injury model. They used scaffolds seeded with fibroblasts adapted from rabbits Achilles tendon for 2 weeks prior to implantation. For the tendon model, results indicated collagen deposition in cell-seeded scaffolds with significant improvement in the mechanical properties from 4 to 12 weeks after implantation. For the ligament-engineering model, authors showed a lack of tissue integration with the bony tunnel attachment with 60% recovery in failure load compared to healthy ligament. Additional research on the chitosan-hyaluronic acid scaffolds has aimed to understand the effects of mechanical stimulation on fibroblasts response [35]. It was found that the application of 90-degree rotations and 5% stretch at 0.5 Hz was associated with increased expression of fibromodulin, and collagens I and III. No further assessment of the overall mechanical influence of the cultured scaffolds under dynamic conditions was made.

Additionally, composite scaffolds made from extruded, cross-linked bovine type I collagen and chondroitin-6-sulfate were fabricated [36]. The collagen-based constructs underwent a series of cross-linking steps using carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS). Cross-linking was combined with freeze-drying to incorporate a porous chondroitin-6sulfate. The final constructs had open and interconnected porosity with an average pore size of 100 [micro]m and axially aligned collagen fibres. Mechanical assessment showed that the cross-linked composite scaffolds could withstand 61.94 [+ or -] 15.54 N, compared to 11.75 [+ or -] 2.62 N without cross-linking. The maximum strain was shown to be 30.17 [+ or -] 7.17% with cross-linked fibres, while it was 15.40 [+ or -] 3.22% without. Composites with cross-linked fibres presented a tensile strength of 1.55 [+ or -] 0.30 MPa compared to 0.24 [+ or -] 0.08 MPa for non-cross-linked fibres. It is important to mention that scaffold porosity is inversely related to mechanical strength and further studies are required to evaluate the cell response of such construct for application in tendon and ligament tissue engineering.

Gelatin was also utilised to fabricate a composite scaffold for tendon tissue engineering applications. Coelectrospinning of aligned poly-[epsilon]-caprolactone (PCL) and methacrylated gelatin (mGLT) followed by photo-cross-linking was used in the fabrication process [34]. Scaffold films were first produced and then were seeded with ADSC, after which photo-cross-linking of 5 layers using UV radiation was made to produce a multilayered scaffold mimicking the natural tendon structure. In vitro assessment showed biocompatibility of produced composite in which ADSCs were oriented along the longitudinal access of aligned fibre construct and expressed tendon-related markers (scleraxis and tenascin-C) after being stimulated with TGF[beta]-3. Mechanical assessment of cell-seeded cross-linked scaffolds however was far inferior for potential clinical application (details in Table 1).

Other groups have combined collagen type I with silk to improve scaffold properties for tendon applications [37]. Sericin protein was extracted from silk fibres produced by Bombyx mori, and mixed with collagen solution to fill the gaps in the silk fibre net. Furthermore, adherence and mechanical strength were increased though DTH crosslinking. Mesenchymal stem cells derived from human embryonic stem cells (hEC-MSCs) were used in in vitro and in vivo models and showed that the mechanicallyloaded knitted silk/ collagen microsponge scaffolds can induce tenogenic differentiation in mesenchymal stem cells and support tendon regeneration up to 360 days after implantation (Table 1).

In an additional study, the same composite scaffolds were tested with the supplementation of recombinant human stromal cell-derived factor-1 alpha (rhSDF-1 alpha), to the collagen type I sponges [38]. SDF-1 is a chemokine that promotes cell recruitment and enhances tissue regeneration. In a murine Achilles tendon model, the authors showed that this approach permits higher expression of collagen one week after implantation, indicating an accelerated onset of tendon healing. Further, the mechanical properties were marginally higher than in scaffolds without rhSDF-1 alpha treatment.

In order to improve the mechanical properties and tendon regeneration, Chen X. et al. utilised the same composite scaffolds with hEC-MSCs [39]. Cells were genetically engineered to overexpress the Scleraxis (SCX) gene. SCX is a transcription factor identified for its role as a tenocyte marker and upstream regulator of tendon-related genes. In vitro and in vivo results showed enhanced tendon regeneration and better quality neotendon formation, compared to the previous studies with the same scaffolds. Importantly, there was less osteogenic, chondrogenic, and adipogenic differentiation of the SCX-hEC-MSCs compared to nongenetically modified MSCs. Mechanical properties in a murine Achilles tendon eight weeks after implantation were inferior to native tendon tissue. This research highlights that scaffold properties are important for tendon tissue engineering, but factors like cell-types used can influence mechanical and histological results.

3.4. Synthetic and Biological Composite Scaffolds. Others have also focused on incorporating PLGA with silk derived materials for tendon tissue engineering applications [40]. A composite scaffold comprised degummed silk microfibers coated electrospun PLGA. The authors degummed silk fibres to achieve a more efficient sericin removal. This resulted in smoother fibre surfaces and preserved the general mechanical properties of the silk. In vitro studies using rabbit bone marrow-derived MSCs revealed good cell viability depending on the seeding technique applied (single or dual surface seeding) and whether scaffolds had a flat or rolled/ cylindrical morphology. Rolled structures had lower cell proliferation compared to the other constructs but, overall, the electrospun PLGA polymer provided a large surface area for cell proliferation. To increase tenocyte differentiation of bone marrow-derived MSCs, authors reported a modified protocol in which the scaffold has a 1-week release of basic fibroblast growth factor (bFGF) [45]. bFGF was blended with PLGA and bovine serum albumin prior to electrospinning with knitted silk fibres. Results showed that incorporating bFGF was associated with upregulation of tenogenic markers during MSC differentiation. Collagen expression was also increased, and this contributed to improved mechanical properties of the scaffold. The combined effect of growth factor incorporation together with dynamic culturing conditions would be of interest for its effect over MSC differentiation and overall construct incorporation.

Sharifi-Aghdam M. et al. [46] have investigated a composite scaffold consisting of knitted silk coated with electrospun collagen-I/polyurethane nanofibres. The main aim of this approach was to incorporate a polyurethane polymer layer to increase the attachment of collagen to silk fibres. In vitro testing using seeded human fibroblast showed that composite scaffold maintained adequate cellular metabolic activity. Assessment of mechanical properties showed an ultimate stress profile reaching 13.5 [+ or -] 1.5MPa and Young's modulus of 21.7 [+ or -] 4MPa. However, further assessment of seeded constructs for their biocompatibility and cell function was not investigated.

Investigative work showed the incorporation of silk with nanofibres derived either from Polycaprolactone (PCL) or Poly(3-hydroxybutyrate) (P3HB) [41]. The composite scaffold was fabricated through electrospinning process of different polymer material over twisted silk fibroin fibres. The main aim of this approach was to incorporate nanofibrous structures onto the composite scaffold to increase the surface to volume ratio and therefore increase cellular attachment. Authors showed a composite scaffold with no toxic effect of seeded fibroblasts with good cellular viability up to day 3 after seeding. Mechanical assessment of fabricated construct showed a maximum load of 97.6 [+ or -] 11.4 N for silk fibroin/P3HB and 110.5 [+ or -] 6.6 N for silk fibroin/PCL with no statistical difference in between. Authors, however, did not further investigate the effect of cellular functions or matrix production or the effect of cell seeding on associated mechanical properties.

Others have focused on the modification of silk to produce scaffolds for tendon regeneration [42]. Degummed silk fibroin meshes were integrated with electrospun aligned silk fibroin cores [42]. In vitro assessment with rabbit MSCs investigated the effects of aligned fabrication techniques, compared to random fabrication, under static and dynamic culture conditions. Results showed that the effects of mechanical stimulation on MSCs was intensified during culture on aligned silk fibroin scaffolds. The dynamic effect was applied in both translational and rotational movement to mimic in vivo environment. This enhanced cellular proliferation and remodelling, with an overall improvement in mechanical properties. Apart from the material used, presence of the aligned scaffold core was proven to be essential for these positive effects when compared to the same scaffolds without alignment. Several authors have showed a similar effect when topography was introduced to the surface of a polymeric scaffold with improvement in cellular alignment and collagen content as well as the expression of different tendon-related extracellular matrix proteins [43, 50].

Elsewhere, collagen has been combined with various polymers through a range of manufacturing techniques to simulate the heterogeneous nature of tendon tissues. Collagen type I was electrospun with synthetic poly(L-lactideco-caprolactone) at a 10:90 ratio, respectively [47]. Fibres were twisted into nanoyarn to produce 150 [micro]m thick scaffolds. Scaffolds aligned randomly or in nanofibers were tested for porosity, surface morphology, and adhesion of tenocytes. Results showed improved cell proliferation in nanoyarn scaffolds, but with poor mechanical properties not useful for future clinical applicability.

In a follow-up study, tendon-derived stem cells (TDSCs) were harvested from rabbit patellar tendons and seeded onto the fibrous scaffolds [48]. In vitro and in vivo assessment under static and dynamic conditions revealed that the composite scaffolds seeded with TDSCs were a promising means for neotendon formation. Furthermore, mechanical dynamic stimulation of the cell-seeded constructs could significantly promote tendon regeneration compared to static culture conditions.

Sensini A. et al. have investigated an electrospun bundled scaffold containing PLA and collagen type I [51]. This scaffold had sufficient mechanical properties with blends containing PLA/collagen 75:25 reaching a Young's modulus of 98.6 [+ or -] 12.4 MPa as spun bundles and 205.1 [+ or -] 73.0 MPa after 14 days of immersion in PBS. Maximum stress was 14.2 [+ or -] 0.7 MPa as spun bundles and 6.8 [+ or -] 0.6 MPa after 14 days in PBS immersion. Tenocytes were metabolically active with good cellular alignment more on blends containing PLA/collagen 50:50 ratio.

Collagen type I has been integrated also with other composite synthetic polymers blends. Polycaprolactone (PCL)/ collagen and poly L-lactide (PLLA)/collagen were evaluated as potential composite materials for tendon-muscle junction tissue engineering [52]. Triphasic scaffolds were fabricated with regions primarily composed of PCL/collagen in one part, PLLA/collagen on the other part, and a mixture of both composites in the middle. All constructs had good biodegradability and biocompatibility. PCL supported myoblast growth due to its low stiffness profile and the higher stiffness of PLLA encouraged fibroblast proliferation. Scaffolds were manufactured using electrospinning technique combined with glutaraldehyde cross-linking and collagen to increase mechanical strength and cell attachment, respectively. In vitro, scaffolds presented good cell integration, viability, and formation of myotubes as those found in tendon-muscle junctions in vivo. Aligned collagen scaffolds were produced with surface cover having polydioxanone (PDS) nanoplates [32]. Assessment in rabbit Achilles tendon demonstrated the same biocompatibility as the previously tested electrospun collagen scaffolds. Further detailed assessment of the composite with and without PDS showed significant improvement in water uptake and release. Histological evaluation showed good tenocyte alignment, neotendon formation, and an initial increase in the inflammatory cell response. The addition of PDS sheets resulted in decreased peritendinous adhesions with higher numbers of mature tenocytes and increased collagen fibril alignment. Improved mechanical properties of the construct were also observed compared to scaffolds made of collagen fibres only [32, 50].

PLLA was also utilised to fabricate a composite scaffold in which electrospinning process was used to form an aligned nanofibres that was later on cross-linked to chitosan-collagen hydrogel mimicking the extracellular matrix of native tendons [53]. The scaffold was rolled and coated on the outer surface with alginate gel aiming to produce an antiadhesion layer around the construct. The scaffold supported cellular alignment and proliferation of tenocytes with no toxic effect. Additionally, adsorption tests showed significantly less attached proteins on the coated surface compared to the noncoated one. Mechanical assessment of produced scaffold showed no effect of coating process on tensile strength of produced scaffold with an effect on the layer number having a value around 2 MPa for 2-layer coated and uncoated scaffolds whereas it was around 6 MPa for uncoated and 4 MPa for coated 3-layer scaffolds. The degradation profile of the scaffold was also investigated and showed that scaffolds maintained 50% of their substance at 21 days after incubation with PBS containing [10.sup.4] units/ml lysozyme solution.

E.C. Green et al. [54] investigated the fabrication of collagen-I/nanocarbon fibres composite scaffold for potential tendon tissue engineering application. Fibres were made through gel-spinning process with the use of a filling load of 0.5 and 5 wt%. This was followed by fibre elongation at a strain rate of 0.02 mm/s and subsequent glutaraldehyde (GA) crosslinking. Material characterization showed a yielded fibre construct similar to native tendon collagen with enhanced mechanical properties.

3.5. Similarities and Dissimilarities between Tendons and Ligaments. Tendons and ligaments have similar structures with different fundamental properties and functions. Tendons are fibrous inelastic structures that connect muscles to bones within joints while ligaments are fibrous but flexible structures important for supporting bone and cartilage. On a structural level, both connective tissues are dense with variable cellular and proteomic elements. Mechanical analysis of human tendons and ligaments showed that the maximum tensile strength ranges from 4.4 to 660 MPa depending on different locations [55, 56]. The maximum strain of these connective tissues was shown to range between 18 and 30%. Young's modulus was shown to range between 0.2 and 1.5 GPa [57, 58]. Structurally, tendons are predominantly composed of a collagen type I matrix containing tenocytes and tenoblasts. In contrast, ligaments contain glycosaminoglycan and lower levels of collagen compared to tendon tissue, with fibroblasts being the main cellular element (Figure 2) [59, 60]. Kharaz Y. et al. compared the extracellular matrix composition of both, natural and tissue engineered, tendons and ligaments [61]. Results showed that, although tissue engineered constructs share the same composition with the native tissue in variable proportions, fundamental differences exist. Specific proteins, such as asporin and tenomodulin, were limited to tendons, while versican, proteoglycan 4, and SOD3 were ligament-specific (Figure 1). Identifying differences in structural protein expression indicates that tissue engineering approaches should aim to replicate these distinctions at the proteomic level. To date, this has not always been the case, and the terms tendon and ligament are often used interchangeably in the literature. This is predominantly because the two connective tissues exhibit similar functional and mechanical properties [12]. An important concept to remember is that cell source is fundamental in dictating the type of the matrix produced [61]. This highlights the intrinsic cell memory that is different between tendon and ligament. In order to tissue engineer these specific tissues, it is important to consider their functional differences so that biomimicry can be achieved. This will eventually help in enhancing integration and function to be restored when used to replace injured tendon or ligaments. From this, it is clear that although the two tissues share several features, a difference exist.

4. Conclusion and Future Directions

This review summarises the current strategies based on composite biomaterials for tendon and ligaments tissue engineering. This approach represents a way to address the heterogeneous nature shared between tendon and ligaments. Although both structures share similar mechanical properties, their constituting cell-types and extracellular matrix compositions are different. Currently, for tendon tissue engineering, several challenges need to be addressed. First, the necessity for a single standard evaluation identifying the mechanical requirements for successful tendon regeneration is lacking. Crucially, this should incorporate various forms of mechanical assessment (including strain and rotation) with different tissue engineering approaches to mimic the natural environment. This is difficult to achieve and implement because of the heterogeneous nature of tendons. The second challenge is to address and control the response of the surrounding tissue to the implanted scaffold. To date, this has been limited to observation of the intrinsic healing response during in vivo applications. Additionally, most of the studies investigating the use of composite materials for tendon and ligament tissue engineering utilise small animals like rabbits rather than larger models like sheep or horses [62]. Such limitations are based on significant weight and mechanical-related difference between in vivo models and native human tissues. Furthermore, the ability to fix the produced construct in situ (with suture, anchors) and shelf life availability should be considered whenever a model is being tested. In fact the interface scaffold-tendon or scaffold-bone is the place at risk of rupture after implantation rather the scaffolds itself.

The described challenges constitute a potential issue for future clinical application of tissue engineered tendon and ligament solutions. Further research is required on the roles of composite materials in order to mimic the appropriate structural, mechanical, and functional characteristics of tendon and ligaments. While synthetic materials are being used currently in clinical practice for tendon and ligament repairs, it will be essential to mechanically match their properties to native tissue. To mimic the mechanical properties of tendon is particularly important as it would allow faster physiotherapy and therefor reduce scar tissue formation which can evolve to articular stiffness. Moreover, this will enhance integration and healing at injury site. The use of certain growth factors such as basic fibroblast growth factor (bFGF) [45] can also be employed during surgery to enhance tissue integration. However this requires a strict control and regulated environment. Since advanced composite materials can be tailored to match different mechanical properties of native tissue, they can be incorporated with growth factors and employed with and without cells, providing large number of possibilities for their clinical use based on site and extent of injuries.

https://doi.org/10.1155/2018/9868151

Conflicts of Interest

None of the authors have any commercial associations or financial relationships that would create a conflicts of interest with the work presented in this article.

Acknowledgments

This study was funded by Royal Free Charity (Award no. 167275).

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Feras Alshomer (iD), (1,2) Camilo Chaves, (1,3) and Deepak M. Kalaskar (1,4)

(1) UCL Centre for Nanotechnology and Regenerative Medicine, Division of Surgery & Interventional Science, University College London, London NW3 2PF, UK

(2) King Khalid University Hospital Surgery Department 37, Plastic and Reconstructive Surgery Section, Riyadh 11472, Saudi Arabia 3Universite Paris Sud, 63 Rue Gabriel Peri, 94270 Le Kremlin-Bicetre, France

(4) Institute of Orthopaedics and Musculoskeletal Science, Division of Surgery and Interventional Science, University College London, Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK

Correspondence should be addressed to Feras Alshomer; dr.fshomer@gmail.com

Received 6 May 2018; Revised 6 July 2018; Accepted 26 July 2018; Published 7 August 2018

Academic Editor: Amit Bandyopadhyay

Caption: Figure 1: Complex structural hierarchy of tendon. Unique structural hierarchy in which collagen molecules represent the simplest forming structure of tendon with complex arrangement up to tendon fascicles producing the final tendon tissue.

Caption: Figure 2: Comparison between natural and tissue engineered tendon and ligament constructs. Unique cellular and extracellular matrix composition that is different between the two types of tissues is important for future clinical translation of tendon and ligament research. SOD3, superoxide dismutase; MPa, mega-Pascal; GPa, giga-Pascal.
Table 1: Summary of the literature related to the applications of
hybrid biomaterial in tendon tissue engineering. * indicates that
approximate numbers were extracted from supplied figures since no
exact reference values are mentioned in the text. PDS: poly-
dioxanone; SCX, scleraxis; TGF[beta], transforming growth factor
beta; hECS-MSCs, mesenchymal stem cells derived from human embryonic
stem cells; rhSDFl, recombinant human sto
PGA, poly-(lactide-co-glycolide); PCL, poly-caprolactone; bFGF,
basic fibroblast growth factor.

COMPOSITE/HYBRID     MODEL              SCAFFOLD
                                        PROPERTIES

PGA/PLA composite.   In vitro           After implantation
[24]                 (ADSCs)            in Achilles
                     In vivo            tendon,
                     (rabbit)           Tensile strength:
                                        4.88 [+ or -] 8.07
                                        MPa

                                        No report of other
                                        mechanical
                                        properties.

P (LLA-CL)/          In vitro           Young's modulus *
Collagen I. [25]     (Tenocytes)        Nonseeded about
                                        2.2 MPa.

                                        Cell seeded about
                                        3 MPa.

                                        Tensile strength *
                                        Nonseeded about
                                        3 MPa.

                                        Cell seeded about
                                        4.5 MPa.

P (LLA-CL)/          In vitro           Mechanical stress:
Collagen I           (TDSCs)            Dynamic group was
nanoyarn. [26]                          59.58 [+ or -]
                     In vivo            7.81 MPa
                     (mouse)
                                        Static group was
                                        43.18 [+ or -] 6.58
                                        MPa

                                        Control group was
                                        32.43 [+ or -]
                                        5.27% MPa

                                        Young's modulus:

                                        Dynamic group was
                                        51.99 [+ or -] 7.16
                                        MPa

                                        Static group was
                                        34.76 [+ or -]
                                        4.75 MPa.

                                        Control group was
                                        23.30 [+ or -]
                                        3.83 MPa

PLA / Collagen-I     In Vitro           Composite
electrospun          (Tenocytes)        scaffolds were
bundles [27]                            made from blends
                                        containing PLA/
                                        Coll-75/25 (w/w).

                                        Young's modulus
                                        was 98.6 [+ or -]
                                        12.4 MPa

                                        Maximum stress was
                                        14.2 [+ or -] 0.7
                                        MPa

PLA-graphene         In vitro           Young's modulus
nanoplatelets        (fibroblast)       PLA control:
(GNP) and                               3.99 [+ or -] 0.42
PLA-carboxyl-        In vivo            GPa
functionalized
carbon               (mouse)            PLA/ CNT-COOH:
nanotubes                               4.86 [+ or -] 0.47
(CNT-COOH)                              GPa
[28, 29]
                                        PLA/ GNP:
                                        4.92 [+ or -] 0.15
                                        GPa

                                        Tensile strength

                                        PLA control:
                                        59.90 [+ or -]
                                        4.93 MPa

                                        PLA/ CNT-COOH:
                                        72.22 [+ or -]
                                        1.52 MPa

                                        PLA/ GNP:
                                        58.56 [+ or -]
                                        3.99 MPa

PCL/ collagen-       In vitro           Tensile strength
PLLA/collagen        (myoblasts,        was  0.5058
[30]                 fibroblast)        [+ or -] 0.2130
                                        MPa

                                        Maximum strain was
                                        18.49% [+ or -]
                                        8.210

                                        Young's modulus
                                        was 7.339 [+ or -]
                                        2.131 MPa

Aligned PLLA         In vitro           Maximum Force to
nanofiber/Layered    (Tenocytes)        break For uncoated
chitosan-collagen                       2 and
hydrogel/                               3 layers scaffold:
Alginate outer                          7.89 [+ or -] 1.5N
coating. [31]                           and 7.45 [+ or -]
                                        0.3N,
                                        For gel coated 2
                                        and 3  layers
                                        scaffolds:  4.76
                                        [+ or -] 0.23N and
                                        6.49 [+ or -]
                                        0.09N.

                                        No report of other
                                        mechanical
                                        properties.

Electrospun          In vivo            Before
collagen I           (rabbit)           implantation:
nanofiber/                              Maximum load was
collagen                                28.33 [+ or -]
microfiber                              2.19 N.
[32]
                                        Maximum stress was
                                        2.69 [+ or -] 0.47
                                        MPa

                                        Maximum strain was
                                        61.34 [+ or -]
                                        4.71 %

                                        Young's modulus
                                        was  43.81 [+ or -]
                                        4.19 KPa. 60
                                        days after
                                        implantation to
                                        Achilles tendon:
                                        Maximum load was
                                        about 63.72 N.

                                        Maximum stress was
                                        about 9.84 N/mm.

                                        Maximum strain was
                                        about 16.35%

                                        Young's modulus
                                        was about
                                        0.62 N/mm.

Electrospun          In vivo            After 60 days of
collagen I           (rabbit)           implantation  to
nanofiber/                              Achilles tendon:
collagen
microfiber/                             Maximum load * was
PDS sheets. [33]                        about 74.02 N

                                        Maximum stress *
                                        was  about 11.37
                                        MPa

                                        Young's modulus *
                                        was about 0.754
                                        MPa

Core-shell           In vitro           Tensilemodulus for
Collagen type I/     (Tenocytes)        dry membranes was
Glycosaminoglycan.                      about 636 [+ or -]
[34]                                    47MPa to 693 [+
                                        or -] 20 MPa

                                        Tensilemodulus for
                                        hydratedmembranes
                                        was 30MPa.

                                        Tensilemodulus for
                                        dry aligned core
                                        scaffold ranges
                                        from 833 [+ or -]
                                        236 to 829
                                        [+ or -] 165 KPa

Collagen-I/          --                 Mechanical
Nanocarbon                              assessment in wet
fibers [35]                             condition

                                        Young's modulus
                                        was  840 [+ or -]
                                        140 MPa

                                        Tensile strength
                                        was 70 [+ or -]
                                        8MPa

Collagen type I/     In vitro           Maximum stress was
Resilin [36]         (Fibroblasts)      34.63 [+ or -] 9.75 MPa

                                        Maximum strain was
                                        0.21 [+ or -] 0.04 MPa

                                        Young's modulus was
                                        49.48 [+ or -] 10.71MPa

poly-[epsilon]-      In vitro (ADSCs)   Maximum load was about 0.25 N.
caprolactone                            No report of other mechanical
(PCL) and                               properties.
methacrylated
gelatin (mGLT)
[37]

Sericin extracted    In vitro           4 weeks after implantation to
knitted              (hESC-MSCs)        Achilles tendon
silk/cross-linked    In vivo (mouse)    of cell-seeded scaffolds:
collagen type I                         Maximum load was
microsponges [38]                       65.24 [+ or -] 9.58 N

                                        Maximum stress was
                                        6.72 [+ or -] 0.90 MPa

                                        Stiffness was
                                        28.26 [+ or -] 2.95 N/mm

                                        Young's modulus was
                                        34.91 [+ or -] 5.08 MPa

Sericin extracted    In vitro           4 weeks after implantation in
knitted silk/        (fibroblast)       Achilles tendon:
cross-linked         In vivo (rat)
collagen type I                         Maximum load was
microsponges/                           68.5 [+ or -] 18 N
rhSDF-1 alpha [39]
                                        Maximum stress was
                                        7.02 [+ or -] 1.7 MPa

                                        Stiffness was
                                        39.0 [+ or -] 6.6 N/mm

                                        Young's modulus was
                                        45.3 [+ or -] 10.4 MPa

Sericin extracted    In vitro           4 weeks after implantation:
knittedsilk/cross-   (hESC-MSCs)        Young's modulus was
linked collagen      In vivo (mouse)    60.63 [+ or -] 17.6 MPa
type I
microsponges/                           Maximum stress
SCX engineered                          was 8.73 [+ or -] 2.15 MPa
cells [40]
                                        8 weeks after implantation:
                                        Young's modulus was
                                        71.3 [+ or -] 12.4 MPa

                                        Maximum stress was
                                        10.1 [+ or -] 2.2MPa

Knitted silk         In vitro           Maximum stress was
coated with          (Fibroblast)       13.5 [+ or -] 1.5 MPa
electrospun
collagen-I /                            Young's modulus was
polyurethane (PU)                       21.7 [+ or -] 4MPa
nanofibers [41]

Silk coated with     In vitro           Maximum load
Polycaprolactone     (Fibroblast)       97.6 [+ or -] 11.4 N for
(PCL) or                                silk fbroin/P3HB
Poly(3-
hydroxybutyrate)                        110.5 [+ or -] 6.6 N for
(P3HB)                                  silk fibroin/PCL
nanofibers [42]
                                        No report of other
                                        mechanical properties.

Degummed             In vitro (MSCs)    Maximum load for dynamic
Silk-fibroin                            culture condition
meshes/aligned                          Aligned scaffold
Silk-fibroin                            144.44 [+ or -] 5.03
[43]                                    N (7 days)

                                        172.08 [+ or -] 6.28
                                        N (14 days)

                                        Random Scaffold
                                        122.35 [+ or -] 3.67
                                        N (7 days)

                                        138.67 [+ or -] 9.22
                                        N (14 days)

                                        Stiffness
                                        Aligned scaffold
                                        24.33 [+ or -] 1.40
                                        N/mm (7 days)

                                        26.93 [+ or -] 2.40
                                        N/mm (14 days)

                                        Random Scaffold
                                        17.48 [+ or -] 0.93
                                        N/mm (7 days)

                                        23.07 [+ or -] 2.54
                                        N/mm (14 days)

                                        No report of other
                                        mechanical properties.

Knitted PLGA-PLLA    In vitro (BMSCs)   Maximum load
/ coating                               Uncoated 68.4 [+ or -] 5.37
with                                    N.
(1) PCL.
(2) PLGA                                PCL coated 63.1 [+ or -] 4.52
nanofiber. Type I                       N.
collagen [44]
                                        PLGA coated
                                        56.3 [+ or -] 6.61 N.

                                        Collagen coated
                                        59.5 [+ or -] 8.3 N.

                                        Stiffness
                                        Uncoated 9.1 [+ or -] 2.38
                                        N/mm.

                                        PCL coated 4.3 [+ or -] 0.91
                                        N/mm.

                                        PLGA coated 5.8 [+ or -] 0.70
                                        N/mm.

                                        Collagen coated 5.7 [+ or -]
                                        0.48 N/mm.

                                        No report of other mechanical
                                        properties.

PLGA nanofiber/      In vitro (BMSCs)   Maximum load of unseeded
Silk microfiber                         scaffolds
[45]                                    75.3 [+ or -] 4.79 N on day
                                        0, and 61.5 [+ or -] 3.43 N
                                        on day 21

                                        Maximum load of rolled seeded
                                        scaffolds
                                        68.2 [+ or -] 6.72 N
                                        on day 21

                                        Stiffness of unseeded scaffolds
                                        4.8 [+ or /] 0.52 N/mm on day
                                        0, and 5.9 [+ or /] 0.54 N/
                                        mm on day 21

                                        Stiffness of rolled seeded
                                        scaffolds
                                        5.5 [+ or -] 0.30 N/mm
                                        on day 21

                                        No report of other mechanical
                                        properties.

PLGA nanofiber/      In vitro           3 weeks after culture of
bFGF / Silk          (mesenchymal       rolled scaffolds:
microfiber [46]      progenitor cell)
                                        Maximum load
                                        Unseeded scaffolds * were
                                        about 61.5 N

                                        Seeded bFGF-free scaffolds *
                                        were about 68.2 N

                                        Seeded bFGF scaffolds * were
                                        about 82.7 N

                                        Stiffness

                                        Unseeded scaffolds * were
                                        about 5.92 N/mm

                                        Seeded bFGF/free scaffolds *
                                        were about 5.53 N/mm

                                        Seeded bFGF scaffolds * were
                                        about 6.97 N/mm

Alginate /           In vitro           Tensile strength was 235.2
0.1% chitosan [47]   (fibroblast)       [+ or -] 8.5 MPa

                                        Maximum strain was 12.3
                                        [+ or -] 0.3 %

Chitosan /           In vitro           (i) In vitro:
0.1% hyaluronic      (fibroblast)       Tensile strength:
acid                 In vivo (rat)      Before seeding was 213.3
[48]                                    [+ or -] 10 MPa

                                        2 hrs after seeding was
                                        60 [+ or -] 6.7MPa

                                        14 days after seeding was
                                        66.7 [+ or -] 6.8 MPa

                                        28 days after seeding was
                                        65.1 [+ or -] 6.6 MPa

                                        Maximum strain:
                                        Before seeding was
                                        3.2 [+ or -] 0.6 %

                                        (ii) In vivo tendon model:
                                        Tangent modulus for cell-
                                        seeded scaffold:

                                        4 weeks after implantation:
                                        about 58 [+ or -] MPa

                                        12 weeks after implantation:
                                        about 85 [+ or -] MPa

                                        In vivo ligament model:

                                        Maximum load for cell-seeded
                                        scaffold:

                                        12 weeks after implantation:
                                        about 110 [+ or -] 10 N

COMPOSITE/HYBRID     MODEL              CELL/TISSUE
                                        INTEGRATION

PGA/PLA composite.   In vitro           (i) Grossly, the
[24]                 (ADSCs)            implanted cell-
                     In vivo            seeded scaffold
                     (rabbit)           was integrated
                                        with the native
                                        tissue
                                        interference, with
                                        a smooth surface
                                        cord-like shape
                                        with less
                                        noticeable
                                        remaining material
                                        after 45 weeks.

                                        (ii) Tissue
                                        adhesions,
                                        described grossly
                                        to be less
                                        compared to
                                        control.

                                        (iii) Parallel and
                                        more mature
                                        collagen fibers
                                        and longitudinally
                                        aligned cells are
                                        presents than
                                        control.

P (LLA-CL)/          In vitro           (i) Significantly
Collagen I. [25]     (Tenocytes)        higher cell
                                        proliferation in
                                        the nanoyarn
                                        scaffold compared
                                        to other scaffolds
                                        and control.

                                        (ii) SEM showed
                                        spindle-shaped
                                        cell in both
                                        nanoyarn and
                                        aligned nanofiber
                                        scaffold, while
                                        polygonal and
                                        random pattern
                                        cells are found in
                                        the random
                                        oriented fibers.

                                        (iii) Expression
                                        of tendon specific
                                        ECM (type I
                                        collagen, type III
                                        collagen, decorin,
                                        tenascin-C, and
                                        biglycan) was
                                        significantly
                                        higher at 14 days
                                        in the nanoyarn
                                        group.

P (LLA-CL)/          In vitro           TDSCs were used in
Collagen I           (TDSCs)            scaffold seeding
nanoyarn. [26]                          with both dynamic
                     In vivo            and static culture
                     (mouse)            conditions.

                                        In vitro:
                                        (i) TDSCs showed
                                        elongated
                                        fibroblast-like
                                        morphology with a
                                        significant
                                        increase in cell
                                        count in dynamic
                                        group at 14 days.

                                        (ii) More cell
                                        infiltration and
                                        dense matrix in
                                        dynamic group.

                                        (iii) PCR
                                        confirmed Tendon
                                        related mRNA
                                        expression more in
                                        dynamic group.

                                        (iv) Western
                                        blotting showed
                                        significant
                                        increase in the
                                        protein expression
                                        levels of
                                        Collagens I and
                                        III, and tenascin-
                                        C in the dynamic
                                        group.

                                        In-vivo:
                                        (i) Significantly
                                        lower number of
                                        cells at 12 weeks
                                        with greater
                                        matrix deposition
                                        and longitudinal
                                        spindle-shaped
                                        cells in dynamic
                                        group compared to
                                        others.

                                        (ii) Collagen
                                        content was
                                        highest in dynamic
                                        group reaching
                                        77.76 [+ or -]
                                        6.82% of normal
                                        rabbit patellar
                                        tendon (174.31
                                        [+ or -] 13.89
                                        [micro]g/mg).

                                        (iii) Collagen I
                                        expression was
                                        Significantly
                                        higher in dynamic
                                        group.

PLA / Collagen-I     In Vitro           (i) Seeded
electrospun          (Tenocytes)        Tenocytes shown to
bundles [27]                            exhibit good cell
                                        adhesion profile
                                        and more elongated
                                        morphology that
                                        was better over

                                        PLA/Coll-50/50
                                        blends than other.

PLA-graphene         In vitro           (i) Both produced
nanoplatelets        (fibroblast)       scaffolds
(GNP) and                               supported
PLA-carboxyl-        In vivo            fibroblasts
functionalized                          metabolic activity
carbon               (mouse)            and proliferation
nanotubes                               till final
(CNT-COOH)                              assessment point
[28, 29]                                (72 hrs).

                                        (ii) In vivo
                                        assessment showed
                                        lack of any local
                                        or systemic
                                        inflammatory
                                        response using N-
                                        acetylglucosaminidase
                                        (NAG) and nitric
                                        oxide (NO) serum
                                        levels.

                                        (iii) No
                                        associated
                                        hepatotoxicity in
                                        histologic
                                        assessment.

                                        (iv) Histologic
                                        assessment of
                                        explanted scaffold
                                        showed formation
                                        of thin capsule
                                        around the implant
                                        with homogenous
                                        granulation
                                        tissue.

PCL/ collagen-       In vitro           (i) Statistically
PLLA/collagen        (myoblasts,        higher viability
[30]                 fibroblast)        of both myoblast
                                        and fibroblasts in
                                        all regions of the
                                        scaffold.

                                        (ii) Scaffold
                                        could support the
                                        formation of
                                        myotubes that is
                                        essential for
                                        normal muscle-ten
                                        don junction
                                        formation.

Aligned PLLA         In vitro           (i) Alginate
nanofiber/Layered    (Tenocytes)        coating was
chitosan-collagen                       associated with
hydrogel/                               significantly less
Alginate outer                          attached proteins
coating. [31]                           than control.

                                        (ii) Both coated
                                        and uncoated
                                        scaffold maintain
                                        50% of their
                                        substance after
                                        incubation with
                                        PBS containing 104
                                        units/ml lysozyme
                                        solution.

                                        (iii) Alamar blue
                                        and DNA
                                        concentration
                                        assessment showed
                                        high cellular
                                        viability,
                                        metabolic
                                        activity, and
                                        proliferation up
                                        to 7 days after
                                        seeding.

                                        (iv) Seeded
                                        scaffolds were
                                        shown to support
                                        cellular
                                        alignment.

Electrospun          In vivo            In vitro:
collagen I           (rabbit)           (i) Significantly
nanofiber/                              higher cell
collagen                                viability in the
microfiber                              aligned hybrid
[32]                                    scaffold compared
                                        to others.

                                        (ii) SEM and
                                        immunofuorescence
                                        proven superior
                                        alignment of
                                        fibroblasts
                                        together with cell
                                        proliferation with
                                        close cell-to-
                                        cell contact in
                                        the aligned hybrid
                                        scaffold compared
                                        to others.

                                        In vivo:
                                        (i) Significant
                                        increase in the
                                        number, density,
                                        and alignment of
                                        the collagen
                                        deposition with
                                        mature elastic
                                        fibers.

                                        (ii) Significant
                                        increase in the
                                        number and
                                        maturity of
                                        tenoblasts and
                                        tenocytes.

                                        (iii) Significantly
                                        lower
                                        peritendinous
                                        adhesions, muscle
                                        fibrosis, and
                                        atrophy and
                                        inflammatory cells
                                        found in the
                                        treated tendons
                                        compared to
                                        controls.

Electrospun          In vivo            (i) Significantly higher
collagen I           (rabbit)           fibrillogenesis after PDS
nanofiber/                              treatment.
collagen
microfiber/                             (ii) Significantly
PDS sheets. [33]                        higher collagen
                                        fibrils were found
                                        in the PDS treated
                                        scaffolds compared
                                        to the regular
                                        one.

                                        (iii) Significant
                                        increase in the
                                        load to failure
                                        and load to yield
                                        point with PDS
                                        treatment.

                                        (iv) Significantly
                                        improved
                                        peritendinous
                                        adhesions, muscle
                                        fibrosis, and
                                        atrophy scores
                                        that are
                                        comparable in the
                                        PDS treated and
                                        nontreated
                                        collagen scaffolds.

Core-shell           In vitro           (i) Significant
Collagen type I/     (Tenocytes)        increase in the
Glycosaminoglycan.                      number of cells at
[34]                                    day 1 after seeding
                                        in the core-shell
                                        composite compared
                                        to core alone
                                        scaffolds.

                                        (ii) Higher
                                        metabolic activity
                                        observed at day 7
                                        in the core alone
                                        scaffold that is
                                        statistically
                                        significant.

                                        (iii) No
                                        significant
                                        difference in the
                                        metabolic activity
                                        and cell number
                                        between the two
                                        groups at 14-day
                                        period.

Collagen-I/          --                 (i) No cell work
nanocarbon fibers                        was  presented.
[35]

Collagen type I/     In vitro           (i) Human adult fibroblasts
Resilin [36]         (Fibroblasts)      were used in the assessment
                                        process.

                                        (ii)Te authors showed that
                                        addition of resilin and the
                                        use of poly(ethylene glycol)
                                        either tetrasuccinimidyl
                                        glutarate did compromise the
                                        cellular activity.

                                        (iii) Te scaffold produced
                                        managed to support cellular
                                        proliferation and 100%
                                        cellular alignment after 7
                                        days of seeding compared to
                                        80% in collagen control.

poly-[epsilon]-      In vitro (ADSCs)   (i) Tenogenic differentiation
caprolactone                            was induced through
(PCL) and                               differentiation medium having
methacrylated                           DMEM, 2% FBS, P-S, and 10 ng-
gelatin (mGLT)                          ml TGF-[beta]3 for 7  days
[37]                                    after seeding.

                                        (ii) Seeded constructs were
                                        shown to exhibit elongated
                                        morphology as well as
                                        expression of
                                        tenogenicmarkers (scleraxis
                                        and Tenascin-C).

                                        (iii) Stacked scaffold was
                                        shown to have adequate
                                        porosity for cell diffusion
                                        and differentiation.

Sericin extracted    In vitro           In vitro:
knitted              (hESC-MSCs)        (i) Good cell attachment,
silk/cross-linked    In vivo (mouse)    proliferation, and spreading
collagen type I                         at 14 days' period.
microsponges [38]
                                        (ii) Scx., collagen I, and
                                        collagen III expression were
                                        significantly higher in
                                        dynamic group than the
                                        control.

                                        In vivo assessment after 4
                                        weeks of implantation:

                                        (i) Grossly, well-integrated
                                        construct with native
                                        tendons.

                                        (ii) Viable cells are
                                        presents showing spindle-
                                        shaped morphology.

                                        (iii) Significantly higher
                                        collagen I, III, V[alpha]1,
                                        V[alpha]2, and TGF[beta]1 in
                                        cell-seeded scaffold compared
                                        to cell free scaffold.

                                        (iv) Neocollagen was
                                        replacing exogenous one with
                                        more content, and mature
                                        morphology in cell-seeded
                                        scaffold compared to cell free
                                        scaffold.

Sericin extracted    In vitro           In vivo assessment after 4
knitted silk/        (fibroblast)       weeks of implantation:
cross-linked         In vivo (rat)
collagen type I                         (i) More fibroblasts-like
microsponges/                           cells and less inflammatory
rhSDF-1 alpha [39]                      cells are found early after
                                        implantation (4 days, 1
                                        week).

                                        (ii) More organized
                                        continuous collagen fibers are
                                        found with a higher
                                        concentration of collagen
                                        type I.

                                        (iii) Good vascular
                                        components with less
                                        inflammatory cells are
                                        present.

                                        (iv) Significantly higher
                                        expression of Collagens I and
                                        III, decorin in the SDF-1
                                        scaffolds at all assessment
                                        periods.

                                        (v) Large fibrils of Achilles
                                        tendons formed in the SDF-1
                                        scaffolds (41.6 [+ or -] 5.5
                                        nm) compared to control (37.1
                                        [+ or -] 2.9 nm).

Sericin extracted    In vitro           In vitro:
knittedsilk/cross-   (hESC-MSCs)        (i) SCX treatment increases
linked collagen      In vivo (mouse)    collagen I expression with
type I                                  enhanced cell-sheet
microsponges/                           formation.
SCX engineered
cells [40]                              (ii) Decreased capacity of
                                        adipogenic, chondrogenic, and
                                        osteogenic differentiation of
                                        the cells with more tenogenic
                                        differentiation.

                                        In vivo:
                                        (i) Good proliferation and
                                        early matrix deposition in
                                        the SCX cells compared to
                                        control.

                                        (ii) Increased Collagen Ia1,
                                        Ia2, and tendon related
                                        transcription factor Eya2 in
                                        SCX cells compared to
                                        control.

                                        (iii) More fibroblast-like
                                        spindle-shaped cells and
                                        fewer immunogenic cell
                                        infiltrates in the SCX cells
                                        group.

                                        (iv) Higher expression of
                                        biglycan in the SCX cells
                                        group, indicating more mature
                                        endogenous collagen.

Knitted silk         In vitro           (i) Human fibroblasts were
coated with          (Fibroblast)       seeded on composite scaffold
electrospun                             to test for viability.
collagen-I /
polyurethane (PU)                       (ii) Assessment was made
nanofibers [41]                         through Alamar Blue assay and
                                        showed that samples with
                                        higher collagen content had
                                        higher cellular viability
                                        profile (COL75/PU25) than
                                        other groups.

Silk coated with     In vitro           (i) Human fibroblasts were
Polycaprolactone     (Fibroblast)       seeded on composite scaffold
(PCL) or                                and showed good cellular
Poly(3-                                 viability and no toxicity
hydroxybutyrate)                        (assessment for 3 days).
(P3HB) nanofibers
[42]

Degummed             In vitro (MSCs)    (i) Highly significant cell
Silk-fibroin                             viability in the aligned
meshes/aligned                          scaffold compared to the
Silk-fibroin [43]                       random one at both dynamic
                                        and static conditions.

                                        (ii) Consistent cellular
                                        proliferation in both aligned
                                        (dynamic and static) and
                                        dynamic random scaffold.

                                        (iii) Higher collagen
                                        deposition in the dynamic
                                        aligned scaffold compared to
                                        the static one and the random
                                        scaffold groups.

                                        (iv) Dynamic culture improved
                                        the histological assessment
                                        of both aligned and random
                                        scaffold with more cell
                                        elongation and matrix
                                        deposition.

                                        (v) Higher expression level
                                        of collagen I, tenascin-C,
                                        and tenomodulin in the
                                        aligned dynamic scaffolds
                                        compared to others.

Knitted PLGA-PLLA    In vitro (BMSCs)   (i) Efficient cell seeding on
/ coating                               PLGA nanofiber coated and
with                                    collagen-coated scaffolds.
(1) PCL.                                (80-89% and 61-69%,
(2) PLGA nanofiber.                     respectively).
Type I collagen
[44]                                    (ii) Significantly higher cell
                                        proliferation between 2nd and
                                        7th culture days of PLGA
                                        nanofiber coated knitted PLGA
                                        scaffolds.

PLGA nanofiber/      In vitro (BMSCs)   (i) Dual surface seeded
Silk microfiber                         scaffolds showed significantly
[45]                                    higher cell proliferation
                                        rates compared to single
                                        surface seeding.

                                        (ii) Increased cell
                                        proliferation between 14 days
                                        and 21 days after culture.

                                        (iii) Rolled-up scaffolds had
                                        nonsignificantly lower cell
                                        proliferation rates.

PLGA nanofiber/      In vitro           (i) Significantly higher cell
bFGF / Silk          (mesenchymal       viability in the bFGF scaffolds
microfiber [46]      progenitor cell)   compared to bFGF-free
                                        scaffolds.

                                        (ii) Significantly higher
                                        collagens I and III,
                                        fibronectin, and biglycan 14
                                        days after culture in the bFGF
                                        scaffolds compared to bFGF free
                                        scaffolds.

                                        (iii) Significantly higher
                                        collagen content in the bFGF
                                        scaffolds compared to bFGF free
                                        scaffolds by 3rd week after
                                        culture.

Alginate /           In vitro           (i) Significantly lower number
0.1% chitosan [47]   (fibroblast)        of unattached cells in
                                        alginate--chitosan group
                                        compared to polyglactin and
                                        alginate alone.

                                        (ii) Fibroblasts were spread
                                        on the polymer fibers.

                                        (iii) Prominent collagen type
                                        I production 14 days after
                                        culture was
                                        more on the scaffold surface by
                                        immune staining with no clear
                                        visualization of both types II
                                        and III collagen.

Chitosan /           In vitro           In vitro up to 28 days after
0.1% hyaluronic      (fibroblast)       cell seeding:
acid                 In vivo (rat)
[48]                                    (i) Gross observation of
                                        ECM production by
                                        lightmicroscopy.

                                        (ii) Prominent collagen type I
                                        production 14 days after
                                        culture more on the scaffold
                                        surface.

                                        In vivo tendon model:
                                        (i) Type I collagen is seen
                                        only in cell-seeded scaffold.

                                        In vivo ligament model:
                                        None.
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Author:Alshomer, Feras; Chaves, Camilo; Kalaskar, Deepak M.
Publication:Journal of Materials
Date:Jan 1, 2018
Words:10545
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