Implantation of bovine hydroxyapatite and secretome with different oxygen concentration may improve massive bone defect regeneration: An experimental study on animal model

The most widely used biomaterials in the treatment of massive bone defects are allograft bone or metal implants. The current problem is that the availability of allographs is limited and metal implants are very expensive. Mass production of secretome can make bone reconstruction of massive bone defects using a scaffold more effective and efficient. This study aims to prove bone regeneration in massive bone defects using bovine hydroxyapatite reconstruction with normoxic and hypoxic secretome conditions using collagen type 1 (COL1), alkaline phosphate (ALP), osteonectin (ON), and osteopontin (OPN) parameters. This is an in vivo study using male New Zealand white rabbits aged 6–9 months. The research was carried out at the Biomaterials Center—Tissue Bank, Dr. Soetomo Hospital for the manufacturer of bovine hydroxyapatite (BHA) and secretome BM-MSC culture under normoxic and hypoxic conditions, and UNAIR Tropical Disease Institute for implantation in experimental animals. Data analysis was carried out with the one-way ANOVA statistical test and continued with the Post Hoc test LSD statistical test to determine whether or not there were significant differences between groups. There were significant differences between hypoxic to normoxic group and hypoxic to BHA group at day-30 observation using ALP, COL 1, ON, and OPN parameters. Meanwhile, there is only osteonectin parameter has significant difference at day-30 observation. At day-60 observation, only OPN parameter has significant differences between hypoxic to normoxic and hypoxic to BHA group. Between day-30 and day-60 observation, BHA and normoxic groups have a significant difference at all parameters, but in hypoxic group, there are only difference at ALP, COL 1, and ON parameters. Hypoxic condition BM-MSC secretome with BHA composite is superior and could be an option for treating bone defect.

A review on bovine hydroxyapatite; extraction and characterization

High rate of bone grafting surgeries emphasizes the need for optimal bone substitutes. Biomaterials mimicking the interconnected porous structure of the original bone with osteoconductive and osteoinductive capabilities have long been considered. Hydroxyapatite (HA), as the main inorganic part of natural bone, has exhibited excellent regenerative properties in bone tissue engineering. This manuscript reviews the HA extraction methods from bovine bone, as one of the principal biosources. Essential points in the extraction process have also been highlighted. Characterization of the produced HA through gold standard methods such as XRD, FTIR, electron microscopies (SEM and TEM), mechanical/thermodynamic tests, and bioactivity analysis has been explained in detail. Finally, future perspectives for development of HA constructs are mentioned.

Fabrication and characterization of bovine hydroxyapatite-gelatin-alendronate scaffold cross-linked by glutaraldehyde for bone regeneration

Objectives Alendronate are widely used in the treatment of bone disorders characterized by inhibit osteoclast-mediated bone resorption such as Paget’s disease, fibrous dysplasia, myeloma, bone metastases and osteoporosis. In recent studies alendronate improves proliferation and differentiation of osteoblasts, thereby facilitating for bone regeneration. The disadvantages of this class are their poor bioavailability and side effects on oral and intravenous application such as stomach irritation and osteonecrosis in jaw. Thus, local treatment of alendronate is needed in order to achieve high concentration of drug. Bovine hydroxyapatite-gelatin scaffold with alendronate was studied. Glutaraldehyde was used as cross-linking agent, increase the characteristics of this scaffold. The objectives of this study were to manufacture and characterize alendronate scaffold using bovine hydroxyapatite-gelatin and crosslinked by glutaraldehyde. Methods Preparation of cross-linked bovine hydroxyapatite-gelatin and alendronate scaffold with different concentration of glutaraldehyde (0.00, 0.50, 0.75, and 1.00%). The scaffolds were characterized for compressive strength, porosity, density, swelling ratio, in vitro degradation, and cytotoxicity (the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, shorted as MTT assay). Results Bovine hydroxyapatite-gelatin-alendronate scaffold cross-linked with glutaraldehyde showed lower density than without glutaraldehyde. As glutaraldehyde concentration increased, porosity also increased. Eventually, it reduced compressive strength. Swelling ratio and in vitro degradation was negatively dependent on glutaraldehyde concentration. In addition, the scaffold has a good safety by MTT assay. Conclusions Bovine hydroxyapatite-gelatin-alendronate scaffold was fabricated with various concentrations of glutaraldehyde. The presence of glutaraldehyde on bovine hydroxyapatite-gelatin-alendronate is safe and suitable candidate scaffold for bone regeneration.

The impact of glutaraldehyde on the characteristics of bovine hydroxyapatite-gelatin based bone scaffold as gentamicin delivery system

Objectives Biomaterials are widely used as drug delivery systems targeting bone tissue, such as to treat bone infectious disease. However, the addition of drugs to biomaterials weakens their mechanical properties. Crosslinkers are compounds that improve the mechanical properties of biomaterials. This study aims to determine the effect of glutaraldehyde (GTA) as a crosslinker on the characteristics of bovine hydroxyapatite-gelatin-based bone scaffold with gentamicin as antibiotics (BHA-GEL-GEN-GTA). Methods BHA-GEL-GEN-GTA scaffold with GTA solid content ranging from 0.1 to 1.4 wt% was made by direct compression. The compressive strength test was carried out using autograph. Scaffold degradation test was carried out by dissolving the scaffolds in PBS. Scaffold toxicity was performed by MTT assay using BHK-21 fibroblast cells. Results There was a significant difference in the scaffolds’ compressive strength due to differences in GTA volume. Scaffold crosslinked using GTA with solid content 0.1 and 0.2 wt% in 2 mL solution had higher compressive strength than those in 1 mL solution. Furthermore, GTA with solid content 0.6, 1, 1.2, and 1.4 wt% showed higher compressive strength than those without GTA. Degradation test results showed that GTA increased the percentage of weight loss and swelling of the scaffold. The scaffold exhibited a nontoxic profile in MTT assay. Conclusions GTA with optimum solid content shows great compressive strength, stable swelling profile with low percentage of scaffold’s weight loss, and is considered as nontoxic.

The comparative Micro-CT analysis on trabecular bone density between hydroxyapatite gypsum puger scaffold application and bovine hydroxyapatite scaffold application

Background: Generally, after tooth extraction, trauma is caused by bone damage, which leads to a decreased bone density. Bone damage repair should be conducted using a bone graft containing hydroxyapatite (HA). HA can be synthesised from gypsum puger powder, which is abundant and easy to obtain. Hydroxyapatite gypsum puger (HAGP) was successful with 100% hydroxyapatite purity level. Purpose: To compare the ratio of trabecular bone density in Wistar rats between HAGP scaffold application and bovine hydroxyapatite (BHA) scaffold application. Methods: This study is a laboratory experiment using 6 treatment groups, namely K (-) polyethylene glycol (PEG) 7, K (-) PEG 28, HAGP + PEG 7, HAGP + PEG 28, BHA + PEG 7, and BHA + PEG 28. HAGP scaffold freeze-drying. The rats were anaesthetised intramuscularly, and their left mandibular incisor was removed. The scaffold was applied to the mouse socket, followed by tissue decapitation after 7 and 28 days. The examination was carried out with micro-computed tomography (Micro-CT). Next, statistical analysis using a one-way analysis of variance (ANOVA) test was conducted (p <0.05). Results: The ANOVA test result showed a difference in bone density between the treatment and control groups on days 7 and 28. The Least Significant Difference (LSD) test result revealed that there was no significant difference between K (-) PEG 28 and HAGP + PEG 7 (p=0.133). Nevertheless, there were significant differences between the other groups. Conclusion: Based on the Micro-CT analysis, the trabecular bone density in Wistar rats following HAGP scaffold application is higher than that of BHA scaffold application.

DIFFERENCES OF BONE REGENERATION USING BOVINE HYDROXYAPATITE AND BOVINE HYDROXYAPATITE WITH FREEZE-DRIED PLATELET RICH PLASMA ALLOGRAFT IN BONE DEFECT OF FEMORAL WHITE RABBIT

Background: Bone defects to date have been a significant problem in the Orthopedics field. Hydroxyapatite is a bone graft that is often chosen if it has osteoconductive properties. Platelet-rich plasma (PRP) has a higher platelet concentration than the concentration in normal blood, capable of providing many bioactive molecules in physiological proportions. Hydroxyapatite given freeze-dried PRP is expected to create a graft that can strengthen the matrix while promoting osteoinduction.Methods: This study compares the effects of regeneration on the bone between bovine hydroxyapatite (BHA) and bovine hydroxyapatite with freeze-dried platelet-rich plasma (FD-PRP) as a bone graft in bone defect of the femoral white rabbit. The 12 equal New Zealand white rabbits aged 6-9 months are divided into two groups. Bone defects were made in the lower femoral meta-diaphysis with a diameter of 2.5 mm. The defects were filled with BHA with FD-PRP allograft in the treatment group and BHA in the control group. Both groups will be sacrificed in the third and sixth weeks, then evaluated histologically for microvascular structure, osteoblasts, woven bone, type-I collagen, osteocalcin, alkaline phosphatase, and immunoglobulin G.Results: During the evaluation in week 3 and 6, microvascular structure, osteoblast, and type-I collagen decreased in both groups with insignificant differences (p>0.05). Woven bone, osteocalcin, and immunoglobulin G increased in the treatment group but was not significant (p>0.05). Alkaline phosphatase increased higher in the treatment group, with a considerable difference in the sixth week (p=0.008).Conclusion: The elevation in the production of woven bone, osteocalcin, and alkaline phosphatase at the third and sixth-week evaluations highlight the possibility that administering BHA given FD-PRP may have contributed to the healing of bone defects.