Fabrication of In Situ-Cross-Linked N-Succinyl Chitosan/Oxidized Alginate Hydrogel-Loaded Ascorbic Acid and Biphasic Calcium Phosphate for Bone Tissue Engineering.

Bone tissue engineering is a promising technology being studied globally to become an effective and sustainable method to treat the problems of damaged or diseased bones. In this work, we developed an in situ cross-linking hydrogel system that combined N-succinyl chitosan (NSC) and oxidized alginate (OA) at varying mixing ratios through Schiff base cross-linking. The hydrogel system also contains biphasic calcium phosphate (BCP) and ascorbic acid (AA), which could enhance biological characteristics and accelerate bone repair. The hydrogels' properties were examined through physicochemical tests such as scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), x-ray diffraction (XRD), pore size and porosity measurement, swelling ratio, degradation rate, AA release study, as well as cytocompatibility, including live/dead and cytotoxicity assays. The results revealed that the supplementation of AA and BCP components can affect the physico-mechanical properties of the hydrogel system. However, they exhibited noncytotoxic properties. Overall, the results demonstrated that the hydrogel composed of 3% (w/v) NSC and 3% (w/v) OA (NSC: OA volume ratio is 8:2) loaded with 40% (w/w) BCP and 0.3 mg/mL AA has the potential for bone regeneration.

Implant stability and clinical outcome between implant placement using internal sinus floor elevation with alloplastic bone material grafting and without grafting: A 1-year randomized clinical trial.

OBJECTIVE: To compare implant stability and clinical outcome in implant placement between osteotome sinus floor elevation (OSFE) with biphasic calcium phosphate (BCP) which consisted of 30% of hydroxyapatite (HA) and 70% of beta-tricalcium phosphate (ß -TCP) grafting material and OSFE without using bone grafting material. The research questions is whether the BCP provides any benefit in OSFE or not. MATERIALS AND METHODS: Thirty patients (30 implants) with a single edentulous area of upper premolar or molar were randomly separated into OSFE with BCP (n = 15) and OSFE without grafting (n = 15). The patients were reevaluated 3, 6, 9, and 12 months after implant loading. The clinical assessments (implant stability quotient (ISQ), implant survival-failure rate, and surgical complication) were analyzed. Together with radiographic assessments in 2D (endo-sinus bone gain (ESBG), mean marginal bone change (MMBC)) and 3D (endo-sinus bone gain in CBCT (ESBG-CT)) were evaluated, with a mean follow-up time of at least 12 months of functional loading and prosthetic complication. RESULTS: 20 remaining implants (OSFE with BCP, n = 10; OSFE without grafting, n = 10) were analyzed. Mean ISQ was 79.18 ± 3.43 in 1-year follow-up (ISQ; OSFE with BCP = 78.72 ± 3.46, OSFE without grafting = 79.65 ± 3.52). ISQ in both groups increased steadily without significant differences in each follow-up. (p = 0.56). In radiographic evaluation, at 6-, 9-, and 12-month, OSFE without grafting group showed statistically significant lower MMBC (p < 0.05). The 1-year clinical results showed that 2 implants failed in OSFE with BCP, and 1 implant failed in OSFE without grafting. CONCLUSIONS: Graft material "BCP" (HA30:TCP70) coupled with OSFE presents no extraordinary benefit in implant stability, clinical and radiographic outcome in 1-year follow-up. CLINICAL RELEVANCE: Clinically, OSFE with grafting materials provides no additional benefit. CLINICAL TRIAL REGISTRATION NUMBER: TCTR20210517008 (date of registration: May 17, 2021).

A biphasic calcium phosphate/acylated methacrylate gelatin composite hydrogel promotes osteogenesis and bone repair.

PURPOSE/AIM: Bone defects caused by trauma, tumors, congenital malformation, or inflammation are very common in orthopedics. In recent years, mimicking the composition and structure of natural bone tissue has become a hot topic in biomaterial research, with the aim of developing an ideal biomaterial for bone defect transplantation. Here, the feasibility of a biphasic calcium phosphate (BCP)/acylated methacrylate gelatin (GelMA) composite hydrogel to repair bone defects was evaluated in vitro and in rats. MATERIALS AND METHODS: The biocompatibility of a biphasic calcium phosphate (BCP)/acylated methacrylate gelatin (GelMA) composite hydrogel was evaluated by cytoskeleton staining, live/dead cell staining and cell proliferation assays. The in vitro osteogenic activities of the composite hydrogel were evaluated by alkaline phosphatase and alizarin red staining, as well as osteogenic gene expression analysis at both transcript and protein levels. The in vivo bone repair activities were evaluated using the rat skull defect model. RESULTS: The BCP/GelMA composite hydrogel displayed excellent biocompatibility and promoted osteogenesis of bone marrow mesenchymal stem cells in vitro. In addition, the BCP/GelMA composite hydrogel markedly promoted new bone formation in the rat skull-defect model. CONCLUSIONS: BCP/GelMA composite hydrogel may be an effective artificial material for bone tissue engineering.

Use of biphasic calcium phosphate versus demineralized bone matrix: retrospective clinical and CT analysis of posterolateral fusion results.

PURPOSE: Bone graft extenders have been developed to prevent donor site morbidity associated with iliac crest bone graft, but few studies compared the efficacy of various substitutes. Our purpose was to determine fusion rate and clinical outcome in patients undergoing lumbar arthrodesis using demineralized bone matrix (DBM) and biphasic calcium phosphate (BCP). METHODS: Patients with degenerative spondylolisthesis undergoing one-level or two-level arthrodesis of lumbar spine were retrospectively reviewed. Two treatment groups placed either BCP or DBM, in addition to local autograft in lumbar posterolateral space. Three-dimensional CT exam and dynamic flexion-extension radiographs at postoperative 2-year were assessed for posterolateral fusion status and pain scale and Oswestry Disability Index (ODI) for clinical outcome. RESULTS: Of the 148 patients reviewed (including 23 in one- and 58 patients in two-level in BCP group, and 47 in one- and 20 patients in two-level in DBM group), no significant differences were found in terms of age, sex, BMI, smoking, diabetes, steroids, number of level fused, non-union rate or revision surgery between BCP and DBM groups. Significantly improved pain scale of back and leg and ODI were found in both groups postoperatively without group difference. We found a comparable fusion rate in one-level surgery (100% versus 93.6%) and a superior fusion rate of BCP group in two-level surgery (98.3% versus 80.0%, p = 0.01). CONCLUSION: Being a bone graft extender without osteoinductive property, with local autograft, BCP is comparable to DBM for one- and superior for two-level fusion. No significant difference was found in clinical outcomes.

A Histologic, Histomorphometric, and Immunohistochemical Evaluation of Anorganic Bovine Bone and Injectable Biphasic Calcium Phosphate in Humans: A Randomized Clinical Trial.

Following trauma, chronic periapical process, or tooth extraction, a large loss of bone volume is noticed during the healing process. To facilitate the placement of dental implants, various surgical procedures are used for an optimal alveolar ridge profile, while maintaining adequate bone dimensions. The main aim of this study was to determine the healing ability (histologically and immunohistologically) of alveolar bone defects during augmentation with two different biomaterials: injectable biphasic calcium phosphate (BCP) and anorganic bovine bone (ABB). Thirty-eight subjects were randomly divided into two groups. The first group received the tested bone substitute biomaterial (BSB), i.e., BCP (maxresorb inject®), and the second group received an alternative to the gold standard, i.e., ABB (Bio-Oss®). The histopathological, histomorphometric, and immunohistochemical analyses gave comparable results for these bone substitute materials in terms of newly formed bone: (BCP: 39.91 ± 8.49%, ABB: 41.73 ± 13.99%), residual biomaterial (BCP: 28.61 ± 11.38%, ABB: 31.72 ± 15.52%), and soft tissue (BCP: 31.49 ± 11.09%, ABB: 26.54 ± 7.25%), with no significant difference found between the groups (p < 0.05, t-test), proving that BCP is equally suitable and successful for alveolar bone regeneration.

Addition of Synthetic Biomaterials to Deproteinized Bovine Bone Mineral (DBBM) for Bone Augmentation-A Preclinical In Vivo Study.

(1) Aim: To investigate the effect of synthetic bone substitutes, α-tricalcium phosphate (α-TCP) or bi-layered biphasic calcium-phosphate (BBCP) combined with deproteinized bovine bone mineral (DBBM), on bone formation. (2) Methods: Thirty critical size defects were randomly treated with the following five different treatment modalities: (1) negative control (NC, empty), (2) DBBM, (3) α-TCP + DBBM (1:1), (4) BBCP 3%HA/97%α-TCP + DBBM (1:1), and (5) BBCP 6%HA/94%α-TCP + DBBM (1:1). The samples, at four weeks post-surgery, were investigated by micro-CT and histological analysis. (3) Results: A similar level of new bone formation was demonstrated in the DBBM with α-TCP bone substitute groups when compared to the negative control by histomorphometry. DBBM alone showed significantly lower new bone area than the negative control (p = 0.0252). In contrast to DBBM, the micro-CT analysis revealed resorption of the α-TCP + DBBM, BBCP 3%HA/97%α-TCP + DBBM and BBCP 6%HA/94%α-TCP + DBBM, as evidenced by a decrease of material density (p = 0.0083, p = 0.0050 and p = 0.0191, respectively), without changing their volume. (4) Conclusions: New bone formation was evident in all defects augmented with biomaterials, proving the osteoconductive properties of the tested material combinations. There was little impact of the HA coating degree on α-TCP in bone augmentation potential and material resorption for four weeks when mixed with DBBM.

Comparison of Injectable Biphasic Calcium Phosphate and a Bovine Xenograft in Socket Preservation: Qualitative and Quantitative Histologic Study in Humans.

This study is the first histologic evaluation of an injectable biphasic calcium phosphate (IBCP) in humans six months after socket preservation according to the principles of guided bone regeneration. After tooth extraction, the alveolar ridge of 21 patients was augmented with IBCP (maxresorb® inject) in the test group, while 20 patients in the control group received a bovine xenograft (BX) (cerabone®). Six months after augmentation, a reentry procedure was performed to collect biopsies of regenerated bone for qualitative and quantitative histologic analysis. A total of 20 biopsies were taken for analysis. Qualitative histologic analysis showed complete integration of the biomaterial and no inflammatory tissue reaction, indicating the biocompatibility of the bone grafts and the surrounding tissue in both groups. Histomorphometric analysis showed comparable results in terms of newly formed bone (IBCP: 26.47 ± 14.71%, BX: 30.47 ± 16.39%) and residual biomaterial (IBCP: 13.1 ± 14.07%, BX: 17.89 ± 11.81%), with no significant difference found across groups (p > 0.05, Mann­Whitney U test). Statistical significance between groups was found in the result of soft tissue percentage (IBCP: 60.43 ± 12.73%, BX: 51.64 ± 14.63%, p = 0.046, Mann­Whitney U test). To conclude, IBCP and BX showed good osteoconductivity and biocompatibility with comparable new bone formation six months after alveolar ridge preservation.

Influence of different carrier materials on biphasic calcium phosphate induced bone regeneration.

OBJECTIVES: Biphasic calcium phosphate (BCP) is a bioceramic material successfully used in alloplastic bone augmentation. Despite many advantages, a disadvantage of BCP seems to be a difficult application and position instability. The aim of this study was to determine how different carrier materials influence BCP-induced quantitative and qualitative bone regeneration. MATERIALS AND METHODS: A total of 70 critical size defects were set in the frontal bone of 14 domestic pigs (5 each) and filled randomly with either BCP alone (BCP), BCP in combination with nano-hydroxyapatite (BCP + NHA), BCP embedded in native porcine type I/III collagen blocks (BCP + C), autologous bone (AB), or were left empty (ED). Specimens were harvested after 4 and 8 weeks and were evaluated histologically as well as histomorphometrically. RESULTS: Significantly lowest rate of new bone formation was found in ED (p = < 0.001) and BCP + NHA groups (p = 0.05). After 8 weeks, the highest percentage of new bone formation was observed in the BCP + C group. Fibrous matrix was detected highest in BCP alone. The lowest residual bone substitute material was found in BCP + C after 8 weeks. CONCLUSIONS: BCP-induced bone regeneration is indeed affected by different carrier types. Surface morphology and bioactive characteristics influence osseointegration and new bone formation in vivo. The combination of type I/III collagen seems most suitable for qualitative and quantitative bone regeneration. CLINICAL RELEVANCE: Stabilization of granular bone substitutes using type I/III collagen might be an alternative to granulates alone, indicating excellent volume stability, satisfactory plasticity, and easy application.

Enhanced Bone Regeneration in Variable-Type Biphasic Ceramic Phosphate Scaffolds Using rhBMP-2.

Our aim was to investigate the bone regeneration capacity of powder-type biphasic ceramic scaffold (BCP powder), block-type BCP (BCP block), and collagen-added block-type BCP (BCP collagen) with different concentrations of recombinant human bone morphogenetic protein 2 (rhBMP-2) in an animal model. Four rabbits were assigned to each of the following groups: no graft + rhBMP-2 (0.1/0.2 mg/mL), BCP powder + rhBMP-2 (0.1/0.2 mg/mL), BCP block + rhBMP-2 (0.1/0.2 mg/mL), and BCP collagen + rhBMP-2 (0.1/0.2 mg/mL), i.e., a total of 32 rabbits. Polycarbonate tubes (Φ 7 mm × 5 mm) for supporting scaffolds were fixed into a 7 mm round border. Subsequently, 0.1 mL of rhBMP-2 solutions with different concentrations was injected into the tubes. Both radiological and histomorphometric analyses showed that osteogenesis was not enhanced by increasing the concentration of rhBMP-2 in all groups at both 3 and 6 weeks. Radiological analysis showed that bone formation was higher in the BCP collagen group than in the BCP powder and BCP block groups at both rhBMP-2 concentrations at 3 weeks. rhBMP-2 enhanced bone formation; however, as the concentration increased, bone formation could not be enhanced infinitely. Collagen-added alloplastic graft material may be useful for mediating rapid bone formation in initial stages.

Novel Silver-Functionalized Poly(ε-Caprolactone)/Biphasic Calcium Phosphate Scaffolds Designed to Counteract Post-Surgical Infections in Orthopedic Applications.

In this study, we designed and developed novel poly(ε-caprolactone) (PCL)-based biomaterials, for use as bone scaffolds, through modification with both biphasic calcium phosphate (BCP), to impart bioactive/bioresorbable properties, and with silver nitrate, to provide antibacterial protection against Staphylococcus aureus, a microorganism involved in prosthetic joint infections (PJIs). Field emission scanning electron microscopy (FESEM) showed that the samples were characterized by square-shaped macropores, and energy dispersive X-ray spectroscopy analysis confirmed the presence of PCL and BCP phases, while inductively coupled plasma-mass spectrometry (ICP-MS) established the release of Ag+ in the medium (~0.15-0.8 wt% of initial Ag content). Adhesion assays revealed a significant (p < 0.0001) reduction in both adherent and planktonic staphylococci on the Ag-functionalized biomaterials, and the presence of an inhibition halo confirmed Ag release from enriched samples. To assess the potential outcome in promoting bone integration, preliminary tests on sarcoma osteogenic-2 (Saos-2) cells indicated PCL and BCP/PCL biocompatibility, but a reduction in viability was observed for Ag-added biomaterials. Due to their combined biodegrading and antimicrobial properties, the silver-enriched BCP/PCL-based scaffolds showed good potential for engineering of bone tissue and for reducing PJIs as a microbial anti-adhesive tool used in the delivery of targeted antimicrobial molecules, even if the amount of silver needs to be tuned to improve osteointegration.

Synergistic Effect of Biphasic Calcium Phosphate and Platelet-Rich Fibrin Attenuate Markers for Inflammation and Osteoclast Differentiation by Suppressing NF-κB/MAPK Signaling Pathway in Chronic Periodontitis.

BACKGROUND: Periodontitis is characterized by excessive osteoclastic activity, which is closely associated with inflammation. It is well established that MAPK/NF-kB axis is a key signaling pathway engaged in osteoclast differentiation. It is stated that that biphasic calcium phosphate (BCP) and platelet-rich fibrin (PRF) have significant antiostoeclastogenic effects in chronic periodontitis. OBJECTIVE: We aimed to elucidate the synergetic effect of PRF/BCP involvement of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the mitogen-activated protein kinase (MAPK) signaling pathway in osteoclast differentiation in chronic periodontitis. METHODS: We induced osteoclast differentiation in vitro using peripheral blood mononuclear cells (PBMCs) derived from patients with chronic periodontitis. We assessed osteoclast generation by tartrate-resistant acid phosphatase (TRAP) activity, proinflammatory cytokines were investigated by ELISA and NF-κB, and IKB by immunoblot, respectively. MAPK proteins and osteoclast transcription factors were studied by Western blot analysis and osteoclast transcriptional genes were assessed by RT-PCR. RESULTS: The results showed that the potent inhibitory effect of PRF/BCP on osteoclastogenesis was evidenced by decreased TRAP activity and the expression of transcription factors, NFATc1, c-Fos, and the osteoclast marker genes, TRAP, MMP-9, and cathepsin-K were found to be reduced. Further, the protective effect of PRF/BCP on inflammation-mediated osteoclastogenesis in chronic periodontitis was shown by decreased levels of proinflammatory cytokines, NF-kB, IKB, and MAPK proteins. CONCLUSIONS: PRF/BCP may promote a synergetic combination that could be used as a strong inhibitor of inflammation-induced osteoclastogenesis in chronic periodontitis.

Dimensional changes of the maxillary sinus augmented with a collagenated synthetic bone block or synthetic bone particulates: A pre-clinical study in rabbits.

OBJECTIVE: To compare the efficacy of a collagenated synthetic bone substitute (C-SBS) to a particulated synthetic bone substitute (P-SBS) in volume maintenance and new bone formations in a rabbit sinus model. MATERIALS AND METHODS: Either C-SBS or P-SBS was grafted in both sinuses of 16 rabbits. Four (N = 8) or 12 (N = 8) weeks after the surgery, total augmented volume (TAV) and area (TAA), as well as new bone volume (NBV) and area (NBA), were statistically compared by radiographic and histomorphometric analyses (p < .05). RESULTS: The differences in TAV, NBV, TAA and NBA between C-SBS and P-SBS groups at 4 weeks were not statistically significant. The TAV (267.13 ± 62.08 vs. 200.18 ± 40.32 mm3 ) and NBV (103.26 ± 10.50 vs. 71.10 ± 7.58 mm3 ) in group C-SBS were significantly higher than in group P-SBS at 12 weeks (p < .05). The TAA (19.36 ± 2.88 vs. 14.48 ± 2.08 mm2 ) and NBA (5.43 ± 1.20 vs. 3.76 ± 0.78 mm2 ) in group C-SBS were significantly higher than in group P-SBS at 12 weeks (p < .05). CONCLUSIONS: Collagenated synthetic bone substitute grafted in rabbit sinuses demonstrated more favourable outcomes across all outcome measures compared to P-SBS at 12 weeks.

Comparative Study between Laser Light Stereo-Lithography 3D-Printed and Traditionally Sintered Biphasic Calcium Phosphate Scaffolds by an Integrated Morphological, Morphometric and Mechanical Analysis.

In dental districts, successful bone regeneration using biphasic calcium phosphate materials was recently explored. The present study aimed to perform a comparative study between 3D-printed scaffolds produced by laser light stereo-lithography (SLA) and traditionally sintered biphasic calcium phosphate scaffolds by an integrated morphological, morphometric and mechanical analysis. METHODS: Biphasic calcium phosphate (30% HA/70% ß-TCP) samples, produced by SLA-3D-printing or by traditional sintering methods, were tested. The experimental sequence included: (1) Microtomography (microCT) analyses, to serve as control-references for the 3D morphometric analysis; (2) loading tests in continuous mode, with compression up to fracture, to reconstruct their mechanical characteristics; and (3) microCT of the same samples after the loading tests, for the prediction of the morphometric changes induced by compressive loading of the selected materials. All the biomaterials were also studied by complementary scanning electron microscopy to evaluate fracture regions and surfaces. RESULTS: The characterization of the 3D mineralized microarchitecture showed that the SLA-3D-printed biomaterials offer performances comparable to and in some cases better than the traditionally sintered ones, with higher mean thickness of struts and pores. Interestingly, the SLA-3D-printed samples had a higher ultimate strength than the sintered ones, with a smaller plastic region. Moreover, by SEM observation, it was observed that fractures in the SLA-3D-printed samples were localized in the structure nodes or on the external shells of the rods, while all the traditionally sintered samples revealed a ductile fracture surface. CONCLUSIONS: The reduction of the region of plastic deformation in the SLA-3D-printed samples with respect to traditionally sintered biomaterials is expected to positively influence, in vivo, the cell adhesion. Both microCT and SEM imaging revealed that the studied biomaterials exhibit a structure more similar to human jaw than the sintered biomaterials.

Fabrication and evaluation of 3D printed BCP scaffolds reinforced with ZrO2 for bone tissue applications.

Fused deposition modeling (FDM) is a promising 3D printing and manufacturing step to create well interconnected porous scaffold designs from the computer-aided design (CAD) models for the next generation of bone scaffolds. The purpose of this study was to fabricate and evaluate a new biphasic calcium phosphate (BCP) scaffold reinforced with zirconia (ZrO2 ) by a FDM system for bone tissue engineering. The 3D slurry foams with blending agents were successfully fabricated by a FDM system. Blending materials were then removed after the sintering process at high temperature to obtain a targeted BCP/ZrO2 scaffold with the desired pore characteristics, porosity, and dimension. Morphology of the sintered scaffold was investigated with SEM/EDS mapping. A cell proliferation test was carried out and evaluated with osteosarcoma MG-63 cells. Mechanical testing and cell proliferation evaluation demonstrated that 90% BCP and 10% ZrO2 scaffold had a significant effect on the mechanical properties maintaining a structure compared that of only 100% BCP with no ZrO2 . Additionally, differentiation studies of human mesenchymal stem cells (hMSCs) on BCP/ZrO2 scaffolds in static and dynamic culture conditions showed increased expression of bone morphogenic protein-2 (BMP-2) when cultured on BCP/ZrO2 scaffolds under dynamic conditions compared to on BCP control scaffolds. The manufacturing of BCP/ZrO2 scaffolds through this innovative technique of a FDM may provide applications for various types of tissue regeneration, including bone and cartilage.

Stem Cell and Advanced Nano Bioceramic Interactions.

Bioceramics are type of biomaterials generally used for orthopaedic applications due to their similar structure with bone. Especially regarding to their osteoinductivity and osteoconductivity, they are used as biodegradable scaffolds for bone regeneration along with mesenchymal stem cells. Since chemical properties of bioceramics are important for regeneration of tissue, physical properties are also important for cell proliferation. In this respect, several different manufacturing methods are used for manufacturing nano scale bioceramics. These nano scale bioceramics are used for regeneration of bone and cartilage both alone or with other types of biomaterials. They can also act as carrier for the delivery of drugs in musculoskeletal infections without causing any systemic toxicity.

Bone Regeneration Using Adipose-Derived Stem Cells in Injectable Thermo-Gelling Hydrogel Scaffold Containing Platelet-Rich Plasma and Biphasic Calcium Phosphate.

For bone regeneration, a biocompatible thermo-gelling hydrogel, hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) was used as a three-dimensional organic gel matrix for entrapping rabbit adipose-derived stem cells (rASCs). Biphasic calcium phosphate (BCP) ceramic microparticles were embedded within the gel matrix as a mineralized bone matrix, which was further fortified with platelet-rich plasma (PRP) with osteo-inductive properties. In vitro culture of rASCs in HA-CPN and HA-CPN/PRP/BCP was compared for cell proliferation and osteogenic differentiation. Overall, HA-CPN/PRP/BCP was a better injectable cell carrier for osteogenesis of rASCs with increased cell proliferation rate and alkaline phosphatase activity, enhanced calcium deposition and mineralization of extracellular matrix, and up-regulated expression of genetic markers of osteogenesis. By implanting HA-CPN/PRP/BCP/rASCs constructs in rabbit critical size calvarial bone defects, new bone formation at the defect site was successfully demonstrated from computed tomography, and histological and immunohistochemical analysis. Taken together, by combining PRP and BCP as the osteo-inductive and osteo-conductive factor with HA-CPN, we successfully demonstrated the thermo-gelling composite hydrogel scaffold could promote the osteogenesis of rASCs for bone tissue engineering applications.

Recombinant human bone morphogenetic protein (rhBMP)9 induces osteoblast differentiation when combined with demineralized freeze-dried bone allografts (DFDBAs) or biphasic calcium phosphate (BCP).

OBJECTIVES: Recently, recombinant human bone morphogenetic protein 9 (rhBMP9) has been characterized as one of the most osteogenic growth factors among the 15 human BMPs. The aim of the present study was to investigate the effects of rhBMP9 in comparison to the clinically utilized rhBMP2 on in vitro cell behavior when combined with two bone graft materials including demineralized freeze-dried bone allografts (DFDBAs) and biphasic calcium phosphate (BCP). MATERIALS AND METHODS: The absorption and release kinetics of rhBMPs from DFDBA and BCP were investigated by ELISA. Moreover, murine bone stromal ST2 cell behavior was investigated on DFDBA or BCP seeded on (1) graft only, (2) rhBMP2 (10 ng/ml), (3) rhBMP2 (100 ng/ml), (4) rhBMP9 (10 ng/ml), and (5) rhBMP9 (100 ng/ml). The effects of rhBMPs on DFDBA and BCP were assessed for cell adhesion, proliferation, and osteoblast differentiation by alkaline phosphatase (ALP) activity, alizarin red staining, and real-time PCR for genes encoding Runx2, ALP, and bone sialoprotein (BSP). RESULTS: While both BMPs were gradually released from DFDBA and BCP over time, significantly higher adsorption was observed on BCP when compared to DFDBA. Cell attachment and proliferation was higher on BCP with little influence of either rhBMP2/9. Despite rhBMPs having relatively no effect on cell attachment/proliferation, a pronounced and marked effect was observed on osteoblast differentiation for both rhBMP2/9. Interestingly, it was observed that rhBMP9 induced significantly higher ALP activity, alizarin red staining, and messenger RNA (mRNA) levels of ALP and BSP when compared to rhBMP2. Our results also revealed higher differentiation for rhBMP2/9 with BCP when compared to DFDBA most likely as a result of higher growth factor adsorption. CONCLUSION: While both rhBMP2/9 combined with DFDBA or BCP induced osteoblast differentiation, rhBMP9 induced greater osteoblast differentiation when compared to rhBMP2. CLINICAL RELEVANCE: rhBMP9 may be a recombinant growth factor with higher potential to induce new bone formation when compared to rhBMP2. Further in vivo studies are necessary to characterize its regenerative potential in various animal models.

The Effect of Reduced Graphene Oxide-Coated Biphasic Calcium Phosphate Bone Graft Material on Osteogenesis.

This study was conducted to evaluate the effect of biphasic calcium phosphate (BCP) coated with reduced graphene oxide (rGO) as bone graft materials on bone regeneration. The rGO-coated BCP bone graft material was fabricatied by mixing rGO and BCP at various concentrations. The surface charge of rGO-coated BCP was measured to be -14.43 mV, which formed a static electrostatic interaction. Cell viabilities were significantly diminished at higher concentrations of ≥100 µg/mL. The calvarial defects of 48 rats were implanted rGO-coated BCPs at a weight ratio of 2:1000 (rGO2), 4:1000 (rGO4), and 10:1000 (rGO10), repectively. BCP was used as a control group. The micro-CT and histological analysis were performed to evaluate new bone formation at 2 and 8 weeks after surgery. The results showed that the new bone volume (mm³) was significantly higher in the experimental groups than in the control group. Histological analysis showed that new bone areas (%) were significantly higher in the rGO2 and rGO10 than in the control, and significantly higher in rGO4 than in the rGO2 and rGO10. Conclusively, the rGO-coated BCP was found to be effective on osteogenesis and the concentration of the composite was an important factor.

Evaluation of New Biphasic Calcium Phosphate Bone Substitute: Rabbit Femur Defect Model and Preliminary Clinical Results.

Autogenous bone grafting, used to repair bone defects, is limited and the donor site can experience complications. Compared to autogenous bone graft, artificial bones have different porosity, which might make them suitable alternatives to bone grafts. Here, two porous biphasic calcium phosphate bone substitutes, namely Bicera™ and Triosite™, are used in an animal study and clinical practice to find a suitable porosity for implantation. Bicera™ and Triosite™ consist of 60 wt% hydroxyapatite and 40 wt% ß-tricalcium phosphate, with the porosity of Bicera™ (82%) being higher than that of Triosite™ (70%). In the animal study, the implantation procedure was carried out on twenty-four female New Zealand rabbits. 12 weeks after implantation, the new bones were well infiltrated into the Bicera™ and Triosite™ bone grafts. In the clinical study, patients with comminuted fracture, fracture nonunion, or arthrodesis were included in the study of bone substitution with Bicera™. 27 patients underwent fracture fixation treatment. Bone healing of 22.22% (6/27) of patients happened within 3 months after the surgery, and that of 66.67% (18/27) of patients happened within 6 months. These results reveal that Bicera™ has good incorporation with host bone, and that new bone is able to grow within the porous structure, giving it high potential in the treatment of bone defects.

Bone regenerative efficacy of biphasic calcium phosphate collagen composite as a carrier of rhBMP-2.

OBJECTIVES: This study compared the bone regenerative effects of a recombinant human bone morphogenetic protein 2 (rhBMP-2)-loaded collagen-based biphasic calcium phosphate composite (BCPC) and rhBMP-2-loaded biphasic calcium phosphate (BCP). MATERIAL AND METHODS: The in vitro release profiles of rhBMP-2-loaded BCP and BCPC were measured. The animal surgery was performed on ten rabbits. Four 8-mm-diameter circular calvarial defects were made and filled with BCP, BCPC, rhBMP-2-loaded BCP (BMP + BCP) and rhBMP-2-loaded BCPC (BMP + BCPC). The animals were euthanized either 2 or 8 weeks after surgery. RESULTS: The initial burst release of rhBMP-2 was greater for BCP than for BCPC, and both presented a slow release pattern thereafter. In rabbit calvarial defects, the space maintaining capability and graft resorption of all experimental groups did not show statistical differences at 2 and 8 weeks. New bone formation in the rhBMP-2-loaded groups was greater than in the non-loaded groups at both weeks, but the amount of new bone was comparable between both rhBMP-2-loaded groups at both weeks. There was a distinct histologic difference between the BMP + BCP and BMP + BCPC groups at 2 weeks; the new bone formation occurred more in the intergranular spaces and the BCP-to-bone contact was greater in the BMP + BCPC group, but these differences were no longer discernible at 8 weeks. CONCLUSIONS: BCP- and BCPC-loaded rhBMP-2 significantly improved bone regeneration and BCPC led to a dense network of new bone and bone particles during the early healing period. BCPC can therefore be considered as a promising candidate for carrying rhBMP-2.