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Bone Remodeling Around Photochemical Fortified-calcium Silicate Implants in Long Term Rabbit Femur Model.

Introduction

Bone remodeling around implants is a major area of interest in the field of biomaterials science. Response of bone tissue to the placed implant comprises of well-orchestrated set of events leading to either healing or failure depending upon the compatibility of the placed implant. Exploration of the remodeling in the periimplant tissue is both interesting and intriguing for a biomaterial scientist, since the complexity of bone healing is further modified by the presence of implant and the substances delivered from the implant.

Calcium silicate is a well-known compound in the field of bone biomaterials science and thoroughly explored for use as bone biomaterial. Previous investigators have used it as ceramic reinforcement in polymer composites [1]. Reports do exist on its use as bone void filler etc. Due to its low thermal stability, high temperature sintering is not feasible. This limits the use of calcium silicate in heavy load bearing cases. Nevertheless non load bearing regions do support use of calcium silicate implants and bone grafts.

Growth factors added to implants are now a central theme in biomaterials science and tissue engineering applications. They have intended to act as tissue growth modifiers to result in the exected outcome. As reported earlier, difficulty in preparing them and high cost of such factors have been limiting the extensive use of such scaffolds in clinical conditions. Since the phytochemistry has shown promising alternative options, study of influence of such bioactive moieties on proximal bone remodeling gains paramount importance [2]. Such studies around native implants have been reported but not with presence of phytochemicals [3].

Incorporation of phytochemicals in calcium silicate implants has been a problem due to inability to exactly quantify amount of bioactive moiety and evaluate its release profile [4]. The debate on use of crude sterile extracts versus isolated phytochemicals is apparently never ending in the field of biomaterials. Crude extracts have shown synergistic activity of various compounds present in them compared with single isolated moiety. Hence in the current study, crude extracts are being used to evaluate the bone remodeling.

Butea monosperma has been reported to contain osteogenic phytochemicals and in our previous work we have shown in vivo its release in porous polymeric implants [2, 5]. The medicarpin is claimed to be the most potent of osteogenic components present in the extract. Glycirrhiza glabra is a traditional Indian herb commonly used to treat multitude of problems. It has been administered orally for millennia and has a well-regarded place in traditional medicine. It is reported to contain osteogenic moieties [6].

Individual phytochemical monographs do claim the bioactivity of phytochemicals but their incorporation in ceramic scaffold for actual use in bone replacement or regeneration is hitherto unexplored, which is a valuable and indispensible data to acquire prior to clinical translation. This study aims to prepare phytochemical incorporated nano-calcium silicate implants and evaluate bone remodeling around the implants over longer periods of time.

Materials and Methods

Preparation of nano-Calcium silicate

Calcium hydroxide and Sodium silicate was purchased from sigma Aldrich and used as received. Nano-calcium silicate was prepared as reported by McFarlane et al [7]. Brieftly, 0.47 Mol calcium hydroxide was mixed with 0.43 Mol sodium silicate solution and stirred at constant rate for 3 hrs. The precipitate was subsequently filtered, washed using deionized water to remove possible unreacted components and dried at 100[degrees]C to remove moisture, stored in cool dry place until use. This is denoted by CS. Prepared calcium silicate was evaluated in FTIR spectroscopy and XRD for phase purity.

Preparation of phytochemical extracts

Dried bark powder of butea monosperma and root powder of glycirrhiza glabra were purchased from government recognized traditional medicine vendors. The powders were extracted using ethanol (10 gm/100 ml) at 37[degrees]C for 48 hrs, subsequently filtered and stored at 4[degrees]C until use.

Fabrication of implants

The CS powder was measured (0.5 g) and pelleted using hydraulic press at 50 MPa for 5 secs. In case of phytochemical incorporated implants, measured qualtity of extracted was thoroughly mixed with powder using mechanical mixer for 3 mins and pelletized as above. The schema for phytochemical concentration is as follows. The extracts were prepared in different dilutions to contain 1, 10, 50, 100 mg solids/ml concentration. 1 ml was added to 0.5 g of CS powder, mixed, air dried and pelletised as explained. The pellets were designated as follows. While Glycirrhiza glabra containing pellets were named as GG1, GG10, GG50 and GG100 respectively in the order of concentrations, Butea Monsperma containing pellets were named BM1, BM10, BM50 and BM100. Pellet size for in vitro studies was of 1 cm diameter and 3 mm height (0.5g). For in vivo studies, the pellets were of 3 mm diameter and 1 mm long (~0.05 g).

In vitro analysis

In vitro biocompatibility analysis was carried out using rat bone marrow derived mesenchymal stem cells isolated using standard protocols. They were cultured in DMEM (HIMEdia Labs, India) containing 15% Fetal bovine serum (HIMEdia Labs India). Unless mentioned, regularly second passage cells were used during this study. The implants were placed in 24 well plates and seeded with MSCs (2 x [10.sup.4] cells per well) analysed for adhesion, viability at 24 hrs and 48 hrs. Each well contained 2 ml of medium.

Cell adhesion analysis

Sterile implants in 24 well plates were seeded with cells as mentioned above. After 3 hrs, wells were rinsed and added with medium containing 10% alamar blue and incubated for 4 hrs. This medium was retrieved and stored, later analysed for absorbance as reported earlier for alamar blue viability assay.

Cell viability assay

After 48 hrs of seeding, medium of wells were replaced with DMEM containing 10% alamar blue dye and incubated for 4 hrs. Subsequently the medium was retrieved, wells washed with PBS to remove traces of alamar blue, added with normal DMEM. All analyses were done in triplicate and cells grown in empty wells served as control [2].

In vivo implantation

The animal experimentation was conducted with approval of Institutional animal ethics committee and according to NIH guidelines for animal care. 6 months old New Zealand white rabbits were used for the study. The study consisted of 3 groups, containing 3 animals each (9 animals in total). Every animal received 2 implants, one per tibia, resulting in 6 samples per group.

After preoperative cleaning and hair removal, under anesthesia, surgical incision was placed on the skin above proximal portion of tibia. After thorough irrigation, access to tibia was gained by blunt dissection of muscle. Under saline irrigation, surgical drill was used to create bone defect such that the implant is placed in snug fit into the defect created. The area was debrided and implant was placed. Suture was placed around the bone in order to secure the implant from accidental dislodgement. Muscular layer was sutured using resorbable sutures followed by skin suture. The site was appropriately dressed to prevent infection. Post operative care was provided according to NIH guidelines including Gentamycin and Buprenorphine for 5 days.

Polyfluorochrome(PFC) injection

Following PFCs were injected intramuscularly in sequence. Tetracycline (12 mg/Kg body weight) on 7, 14, 21st days; Alizarin red S (30 mg/Kg body weight) on 28, 35 and 42nd days; Calcein (5 mg/Kg body weight) on 48, 56 and 63rd days.

Histology and histomorphometry

After 180 days, the animals were sacrificed using overdose of anesthesia and implant bone complex was retrieved, fixed in absolute ethanol. Processing for hard tissue section was done according to Maat et al [8]. Briefly, the fixed specimens were dried in isopropanol, embedded in acrylic and section was ground using abrasive paper to obtain ground sections. The samples were visualized in epifluorescence microscope (Olympus CX41 with fluorescent attachment) at emission of 450nm. Bone implant contact was analyzed as proportion of actual contacting area to total contacting area using NIH-ImageJ ver 2 software.

Statistical analysis

All data are presented as (mean [+ or -] standard deviation). Statistical analysis was performed using one way ANOVA or student t-test when appropriate. Differences at p < 0.05 were considered to be statistically significant.

Results and Discussion

Preparation of nano-Calcium silicate

Prepared calcium silicate was evaluated in FTIR spectroscopy and

XRD for phase purity

The FTIR spectrum of prepared calcium silicate shows typical functional groups as reported by Yu et al., confirming the purity [9] (Fig. 1). The XRD spectrum typically demonstrates the phase purity of calcium silicate as tobermorite corresponding to [0,2,0] and [2,2,2] at 29 and 30 degrees respectively [10].

Cell culture analysis

The in vitro analysis is carried out to determine the best group to be used in the in vivo studies. Adhesion assay is basically a measure of live cells present after given period subsequent to seeding on scaffolds. In current study adhesion assay was performed using alamar blue dye and it showed good adhesion of stem cells on the scaffold. Adhesion of cells and subsequent spreading depend on recognition of scaffold by cells' membrane, following which fibronectin is actively synthesised in the cells. This phenomenon can be altered in the presence of phytochemicals or other bioactive substances. In this case, addition of phytochemicals has improved cell adhesion as shown in figure 2. The improvement in adhesion had linear dependence on photochemical content till it reached an optimum level and later formed plateau--giving a sigmoid picture of graph. This states that improvement by phytochemicals has a limit.

Cell viability is the function of total surviving cells, after they have survived the seeding procedure. The stress whatsoever the cell undergoes during detachment and seeding definitely reflects in cell viability. Presence of bioactive agents can improve the cell survival and hence viability on BM and GG are better than CS groups. In earlier studies on phytochemicals such phenomena has been observed. [2, 5]

It is observed that GG50 and BM50 showed good adhesion and proliferation and beyond that concentration the activity did not improve. Hence they were selected for in vivo studies. This phenomenon is demonstrated earlier by Muthusami et al [11]. Precisely the only till a particular concentration, bioactivity is related to it linearly, beyond which the bioactivity remains constant.

Polyfluorochrome(PFC) injection based histomorphometry

Initial sequence of events following bone injury is inflammation, callus formation and osteoid deposition followed by maturation. In case of bone implant placement, the initial healing response remains the same except for the deposition around the placed implant. Initial 3 week period is hence expected to show diffuse patterns of tetracycline(TC) lines sandwiched between normal bone and placed implant. In case of unaltered healing this will remain as it is even after 6 months, despite future PFC injections. This scenario is well observed in control group implants (CS) (Fig. 3, 4, 5). In case of GG50, the zone of TC lines is shortened indicating earlier completion of inflammatory phase and possible 'limiting' of the radius of inflammation. Whereas, BM50 shows inconspicous TC zone which clearly indicates earlier completion of inflammatory phase and continued remodeling in the later periods.

In the 4-6 weeks period, (period of Alizarin Red S injections(ARS) the healing is expected to be complete in close proximity to the implant, establishing histologically assessible bone implant contact. Hence ARS if seen near implant strongly indicates establishment of BIC. In none of the samples such phenomena is seen because of modification of healing process by implants. In CS and GG50 group, the ARS is seen in farther from implant indicating healing is not happening near the implant. In contrast BM implants shed proximal deposition of ARS indicating active chemotaxis and osteogenesis.

In the 7-9 weeks period (Period of Calcein injection (CL)), in normal healing scenario, no remodeling should be seen only incremental and reversal lines need to be observed. If CL is present, it indicated prolonged osteogenesis. CS samples show no calcein indicating resting of bone deposition--completed healing. Profuse lines of calcein in GG25 indicate proceeding osteogenesis. Well defines lines in BM25 shows early maturation and continuing osteogenesis, probably a result of inhibited osteoclastogenesis. This effect is demonstrated in vitro in BM by Maurya et al. [5].

In summary while healing around the CS is satisfactory as known, incorporation of GG and BM has positively modified the inflammatory and remodeling processes of the peri-implant bone. While both GG and BM has shown early completion of inflammatory phase, BM showed longer and sustained osteogenic activity compared to GG.

Bone-implant contact and distance of proximity

The distance between formed bone and the implant is a major factor in clinical translation of bone implant materials and is bound to reduce in case of chemotaxis. The migration of cells and higher turnover rate are characteristic of local chemotactic activity and hence has been measured in this study. While CS showed mean BIC as 75 [+ or -] 2%, both GG50 and BM50 showed 81 [+ or -] 1% respectively.

Conclusion

The study has thrown valuable light in the field of bone tissue engineering using phytochemicals. Both BM and GG are shown to improve peri-implant healing and improved osseointegration of bioceramics. BM is shown to produce sustained osteogenic activity.

In future, BM can be incorporated in to bone tissue engineering scaffolds to produce more successful clinical outcome.

Received 1 October 2017

Accepted 25 October 2017

Published online 30 April 2018

References

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[3.] S. Allegrini Jr, E. Rumpel, E. Kauschke, J. Fanghanel, B. Konig Jr., Hydroxyapatite grafting promotes new bone formation and osseointegration of smooth titanium implants. Ann. Anat. 2006; 188 : 143-151

[4.] A.M.A.Sasidharan, Y Chen, D. Saravanan, K.M. Sundram, L. Yoga Latha, Extraction, isolation and characterization of bioactive compounds from plants' extracts. Afr. J. Tradit. Complement. Altern. Med, 8, 1-10. (2011)

[5.] R. Maurya, D.K. Yadav, G. Singh, B. Bhargavan, P.S. Narayana Murthy, M. Sahai, M.M. Singh, Osteogenic activity of constituents from Butea monosperma, Bioorg. Med. Chem. Lett., 19, 610-3 (2008).

[6.] C. Simmler, G.F. Pauli, S.N. Chen, Phytochemistry and biological properties of glabridin. Fitoterapia, 90, 160-184 (2013)

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[8.] G. Maat Jr., R. P.M. Van Den Bos, M. Aarents, Manual preparation of ground sections for the microscopy of natural bone tissue: update and modification of Frost's 'rapid manual method'. Int. J. Osteoarchaeol, 11, 366-374 (2001)

[9.] Ping Yu, R. J. Kirkpatrick, B. Poe, P. F. McMillan, X. Cong. Structure of calcium silicate hydrate (C-S-H): Near-, Mid-, and Far-infrared Spectroscopy. J. Am. Ceram. Soc, 82, 742-48 (1999)

[10.] S. Grangeon, F. Claret, Y. Linard, C. Chiaberge. X-ray diffraction: a powerful tool to probe and understand the structure of nanocrystalline calcium silicate hydrates. Acta Cryst., B69, 465-473 (2013)

[11.] S. Muthusami, K. Senthilkumar, C. Vignesh, R. Ilangovan, J. Stanley, N. Selvamurugan, et al. Effects of Cissus quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells. J. Cell Biochem., 112, 1035-1045 (2011).

R Narasimha Raghavan *, G. Vignesh, N. Mohana, D. Sivaraman, P.S. Pradeep

Centre for Laboratory Animal Technology and Research, Sathyabama University, Chennai 600119

* Coresponding author.

E-mail address: raghav14rn@hotmail.com (Dr. R. Narasimha Raghavan)

Caption: Figure 1: FTIR and XRD Spectra

Caption: Figure 2: In vitro studies

Caption: Figure 3: CS group and histomorphometry

Caption: Figure 4: CS-GG and histomorphometry

Caption: Figure 5: CS-BM and histomorphometry
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Title Annotation:Original Article
Author:Raghavan, R.Narasimha; Vignesh, G.; Mohana, N.; Sivaraman, D.; Pradeep, P.S.
Publication:Trends in Biomaterials and Artificial Organs
Date:Jan 1, 2018
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