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Controlled Processing of Functionalized Biomineralized Bone-like Scaffolds Through Ribose Reinforcement Obtained by Biomimetic Strategies for Bone Augmentation.


Developing, biomimetic, bone-like scaffolds with analogous mechanical and resorption properties of the native bone is of clinical importance. An approach to reconciliate this clinical demand is to tailor scaffolds with osteogenic cues and collectively strengthen the scaffold mechanical properties through crosslinking reactions [1]. Apart, from the plethora of crosslinking strategies, ribose crosslinking is a novel, low-cost coupling agent used to obtain highly stable, solid and durable scaffold for hard tissue engineering application.

This study exploits, the opportunity of using ribose as a cross-linking agent to develop magnesium-doped-hydroxyapatite collagen (MgHA/Coll) scaffolds through a bioinspired biomineralization process. To this aim, the crosslinking mechanism in terms of preribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) was investigated.

The study result explicates that, scaffolds exhibited good swelling behaviour with controlled resistance to enzymatic degradation. The fast-swelling response is the characteristic feature attributed to the high hydrophilicity and porosity of MgHA/Coll scaffolds and the enhanced enzymatic resistivity is due to newly formed crosslinks between collagen molecules due to ribose interaction helps in masking of collagenase recognition sites [2].

Morphological features such as porosity (> 80%), pore size (0-200 gm) and presence of amorphous mineral phase was evident through SEM. The scaffolds revealed to be highly fibrous with micro-architectures of very small nanocrystals closely bound to the collagen fibres thereby giving the material a highly porous structure with micro/macro porosities. ICP results demonstrated the magnesium substitution of calcium and the Ca/P ratio was within the range of (1.45-1.60) which construes the typical low crystalline apatite range which is mainly due to the presence of [HPO2.sup.-4] ions [3].

The FTIR spectral changes confirms the nucleation of [Mg.sup.2+] ions in the collagen template which is exceptionally amorphous and non-stoichiometric with evidence of high level biomimicry analogous to the natural bone mineral and the active interaction and presence of ribose as crosslinker within the collagen and MgHA heteromolecular networking as seen in Fig. 1.A. The XRD exhibited a low crystalline pattern with large broadening of reflections which shows that during the nucleation process, the nano-sized dimensions of crystallites were growing along the collagen fibrils with preferential r-axis orientation as seen in Fig. 1.B.

The TGA affirmed the nucleation of mineral phase with evidence showing that high temperature crosslinking reaction does not disturb the nucleation of the mineral phase and also helps in producing an equivalent reaction yield as observed in Fig. 1.C [4]. In addition, a favourable mechanical behaviour in response to compressive force was achieved. The performed mechanical characterization shows that the presence of ribose did have a significant effect in ameliorating the compressive modulus of the MgHA/Coll scaffolds as depicted in Fig. 1.D.

The preliminary cell culture experiments represented a satisfying biocompatibility with good cell adhesion, proliferation and colonization. This preliminary in-vitro study was performed to assess the functional utility of this novel, low-cost crosslinker in MgHA/Coll scaffolds in terms of cell-material interaction as seen in Fig. 2 With the increase in culture time there was increase in cell density and cells appeared to be homogenously distributed in both the scaffold surfaces as shown in Fig. 2.A.

From the cytoskeleton structure staining, the proper presence of extended actin filaments with cell nuclei manifests the cell survival with rational cell-material interaction under the influence of ribose in both pre and post glycated samples as seen in Fig. 2.B. The detailed cell morphology, as observed from the SEM analysis shows the strong scaffold colonization with the characteristics osteoblast morphology without any abnormal alterations as depicted in Fig. 2.C. The cell proliferation reported as percentage of live cells, demonstrated a high level of cell viability as the culture time prolonged as illustrated in Fig. 2.D.

Overall, the presence of ribose can collectively strength the matrix stiffness which greatly influences cell proliferation and adhesion. In addition, the presence of HA within the collagen networking has greatly helped in obtaining good surface properties (suitable roughness) and opportune hydrophilicity which greatly improves the biocompatibility of the scaffolds. This in- vitro study strongly supports this hypothesis, that the minimum concentration of ribose with nominal ratio of HA helps in obtaining highly desirable crosslinking with opportune surface roughness that collectively benefits the overall biocompatibility of these scaffolds [5]. To conclude, a proof of concept of ribose being a cost-effective, safe and effective crosslinker has been demonstrated. Therefore, the ribose glycation of collagen can serve as a value tool to study, (i) altered tissue mechanical properties due to glycation, (ii) cell behaviour in response to glycation, (iii) as in-vitro model to study extra cellular matrix changes in age-related bone pathologies. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering.


[1.] Sprio S, Ruffini A, Valentini F, D'Alessandro T, Sandri M, Panseri S, Tampieri A. Biomimesis and biomorphic transformations: new concepts applied to bone regeneration. J Biotechnol. 2011,20; 156(4):347-55.

[2.] Charulatha V, Rajaram A, Influence of different crosslinking treatments on the physical properties of collagen membranes, Biomaterials. 24(5) (2003) 759-67.

[3.] Minardi S, Corradetti B, Taraballi F, Sandri M, Van Eps J, Cabrera FJ, Weiner BK, Tampieri A, Tasciotti E, Evaluation of the osteoinductive potential of a bio-inspired scaffold mimicking the osteogenic niche for bone augmentation, Biomaterials. 62 (2015) 128-37.

[4.] Ramirez-Rodriguez GB, Delgado-Lopez JM, Iafisco M, Montesi M, Sandri M, Sprio S, Tampieri A, Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration, J Struct Biol. (2016)

[5.] Krishnakumar GS, Gostynska N, Campodoni E, Dapporto M, Montesi M, Panseri S, Tampieri A, Kon E, Marcacci M, Sprio S, Sandri M. Ribose mediated crosslinking of collagen- hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies. Mater Sci Eng C Mater Biol Appl. 2017,1; 77:594-605.

K. Gopal Shankar

Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, Erode, Tamilnadu, India

Caption: Figure 1: A. FTIR peaks, B.XRD peaks, C. TGA curves, D. Stress-strain curves of pre and post ribose glycation of MgHA/ Coll scaffolds

Caption: Figure 2: A. Live/Dead stain, B. Actin/DAPI stain, C. SEM morphology, D. Cell viability of ribose glycation of MgHA/ Coll scaffolds
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Title Annotation:Original Article
Author:Shankar, K. Gopal
Publication:Trends in Biomaterials and Artificial Organs
Date:Jan 1, 2018
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