Osteoinductive Biphasic Calcium Phosphate Research Articles

One-step co-doping of ZnO and Zn2+ in osteoinductive calcium phosphate ceramics with synergistic antibacterial activity for regenerative repair of infected bone defect

How to endow bone grafts with long-term antibacterial activity and good bone regenerative ability to achieve the regenerative repair of infected bone defects has been the focus of the clinical treatment of osteomyelitis. The present study introduced a novel one-step route to realizing the co-doping of zinc oxide (ZnO) and zinc ion (Zn2+) in biphasic calcium phosphate (BCP) ceramics to utilize their synergistic antibacterial. Compared with the conventional BCP ceramics (BCP-Ca), the ZnO/Zn2+ co-doping ones (BCP-Zn) possessed strong antibacterial ability on E. coli and S. aureus as well as stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) effectively. The synergistic antibacterial mechanism of ZnO and Zn2+ was also investigated. BCP-Zn showed excellent osteoinductivity and angiogenesis at three months postoperatively in the canine intramuscular implantation model. Moreover, BCP-Zn exhibited excellent anti-infective ability and bone regenerative repair compared to BCP-Ca and control groups in the infected bone defect model of rat femur. Collectively, these findings suggest that the simultaneous introduction of ZnO/Zn2+ could have immense potential to expand the application of osteoinductive BCP ceramics in the regenerative repair of infected bone defects.

Optimal regenerative repair of large segmental bone defect in a goat model with osteoinductive calcium phosphate bioceramic implants

So far, how to achieve the optimal regenerative repair of large load-bearing bone defects using artificial bone grafts is a huge challenge in clinic. In this study, a strategy of combining osteoinductive biphasic calcium phosphate (BCP) bioceramic scaffolds with intramedullary nail fixation for creating stable osteogenic microenvironment was applied to repair large segmental bone defects (3.0 cm in length) in goat femur model. The material characterization results showed that the BCP scaffold had the initial compressive strength of over 2.0 MPa, and total porosity of 84%. The cell culture experiments demonstrated that the scaffold had the excellent ability to promote the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs). The in vivo results showed that the intramedullary nail fixation maintained the initial stability and structural integrity of the implants at early stage, promoting the osteogenic process both guided and induced by the BCP scaffolds. At 9 months postoperatively, good integration between the implants and host bone was observed, and a large amount of newborn bones formed, accompanying with the degradation of the material. At 18 months postoperatively, almost the complete new bone substitution in the defect area was achieved. The maximum bending strength of the repaired bone defects reached to the 100% of normal femur at 18 months post-surgery. Our results demonstrated the good potential of osteoinductive BCP bioceramics in the regenerative repair of large load-bearing bone defects. The current study could provide an effective method to treat the clinical large segmental bone defects.

Evaluation of osteoinductive calcium phosphate ceramics repairing alveolar cleft defects in dog model.

Alveolar cleft repair is an important step in the sequence of treatments for cleft lip and palate. Intrinsically osteoinductive materials have been the subject of research interest. The aim of this study was to explore the use of osteoinductive biphasic calcium phosphate (BCP) ceramics to repair alveolar cleft defects in dogs. We prepared two kinds of BCP ceramic with different physical characteristics: osteoinductive BCP (OBCP) and non-osteoinductive BCP (NBCP). Bilateral alveolar cleft models were surgically established in dogs. On one side, OBCP was implanted in the defect; on the opposite side NBCP was implanted as a control. The materials were also implanted in the femoral muscles to test their properties at non-osseous sites. The osteogenic ability of materials was evaluated with imaging, spiral CT, histology and fluorescent dye tests. At the muscular implantation sites, new bone formed in all of the OBCP samples, but none in the NBCP samples. Imaging and spiral CT revealed good appearance and continuity of the alveolar cleft postoeration, with normal eruption of the bilateral permanent teeth in the groups. Histological and fluorescent dye testing revealed new bone formation in both groups in situ. However, earlier osteogenesis initiation and bone remodeling were superior with OBCP. Osteogenic process in the intramuscular samples with OBCP was similar to that seen in situ. Our findings indicated that osteoinductive biphasic calcium phosphate ceramics (OBCP) have superior characteristics in alveolar cleft repair compared with non-osteoinductive calcium phosphate ceramics (NBCP).

Role of biphasic calcium phosphate ceramic-mediated secretion of signaling molecules by macrophages in migration and osteoblastic differentiation of MSCs.

It is known that the inflammatory reaction initiates fracture healing. The aim of this study was to examine whether osteoinductive BCP ceramics could cause macrophages to change their secretion patterns and whether the secreted cytokines could affect migration and osteoblastic differentiation of MSCs. Moreover, the duration of inflammation could be influenced by the local ionic environment and the degradation products of the implant. Our experimental results revealed the correlation between the inflammatory reaction and osteoinductive capacity of BCP ceramic. The ceramic itself and its degradation products can induce macrophages to express and secrete various signaling molecules, which then recruit and promote the MSCs to differentiate into osteoblasts. Compared with ionic microenvironment, degradation particles played a more substantial role in this process. Therefore, the appropriate inflammation initiated by BCP ceramic and its degradation products could be essential for osteoinduction by the ceramic. We believe that the present study improves the understanding of the effect of biomaterial-mediated inflammation on MSC migration and differentiation and established a preliminary correlation between the immune system and osteoinduction by biomaterials.

In vitro characterization of an osteoinductive biphasic calcium phosphate in combination with recombinant BMP2

BackgroundThe repair of alveolar bone defects with growth factors and bone grafting materials has played a pivotal role in modern dentistry. Recombinant human bone morphogenetic protein-2 (rhBMP2), an osteoinductive growth factor capable of cell recruitment and differentiation towards the osteoblast lineage, has been utilized in combination with various biomaterials to further enhance new bone formation. Recently, a group of novel biphasic calcium phosphate (BCP) bone grafting materials have been demonstrated to possess osteoinductive properties by demonstrating signs of ectopic bone formation. The aim of the present study was to study the effects of rhBMP2 in combination with osteoinductive BCP bone grafts on osteoblast cell behaviour.MethodsMC3T3-E1 pre-osteoblasts were seeded on 1) control tissue culture plastic, 2) 10 mg of BCP alone, 3) 100 ng rhBMP2, and 4) 100 ng rhBMP2+ 10 mg of BCP and analyzed for cell recruitment via a Transwell chamber, proliferation via an MTS assay and differentiation as assessed by alkaline phosphatase (ALP) activity, alizarin red staining and real-time PCR for osteoblast differentiation markers including Runx2, collagen1, ALP, and osteocalcin (OCN).ResultsrhBMP2 was able to significantly upregulate cell recruitment whereas the addition of BCP as well as BCP alone had no additional ability to improve osteoblast recruitment. Both BCP and rhBMP2 were able to significantly increase cell proliferation at 3 and 5 days post seeding and cell number was further enhanced when rhBMP2 was combined with BCP. In addition, the combination of rhBMP2 with BCP significantly improved ALP activity at 7 and 14 days post seeding, alizarin red staining at 14 days, and mRNA levels of Runx2, ALP and osteocalcin when compared to cells seeded with rhBMP2 alone or BCP alone.ConclusionsThe results from the present study demonstrate that 1) the osteoinductive potential of BCP bone particles is equally as osteopromotive as rhBMP2 on in vitro osteoblast differentiation and 2) BCP particles in combination with rhBMP2 is able to further increase the osteopromotive differentiation of osteoblasts in vitro when compared to either rhBMP2 alone or BCP alone. Future animal testing is further required to investigate this combination approach on new bone formation.

Osteogenic gene array of osteoblasts cultured on a novel osteoinductive biphasic calcium phosphate bone grafting material.

Recently, novel biphasic calcium phosphate (BCP) scaffolds have emerged as a new class of bone grafts with osteoinductive potential demonstrating the ability to form ectopic bone in extra-skeletal sites. The aim of the present study was to perform an osteogenic gene array to target possible genes responsible for eliciting the changes in cell expression responsible for inducing osteoblast differentiation. Human MG63 osteoblast-like cells were seeded for 24h on tissue culture plastic or osteoinductive BCP particles and analyzed for upregulated genes using an osteogenesis super-array. Osteoblast-related genes including those transcribed during bone mineralization, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules were investigated. An upregulation of genes transcribing biglycan (1.7-fold), bone morphogenetic proteins 1, 2, 4, 6, and 7 (1.5-2.1-fold), various collagen isoforms including 1a1, 1a2, 2a1, and 5a1 (1.73-2.72-fold), colony stimulating factor 2 (2.59-fold), fibroblast growth factor receptor 2 (1.79-fold), fibronectin (2.56-fold), integrin alpha 1, 2, and 3 (1.82-2.24-fold), SOX9 (2.75-fold), transforming growth factor beta receptor 2 (1.72-fold), vitamin D (1.89-fold), and vascular endothelial growth factor A and B (2.00, 1.75-fold) were all significantly (p<0.05) increased on BCP particles when compared to control tissue culture plastic. In summary, a number of activated genes were involved in bone formation following osteoblast attachment to BCP particles. The involvement of key chondrogenic genes hints that bone grafts capable of spontaneously inducing ectopic bone formation may implicate endochondral ossification. Further investigations in the triggered pathways involved in the process of ectopic bone formation are necessary to understand the key inductive properties of these novel osteoinductive BCP particles. Novel osteoinductive BCP particles demonstrate a wide range of significant increases over several key molecules implicated in osteogenesis that may be implicated in their ability to form ectopic bone formation.

Addition of a Synthetically Fabricated Osteoinductive Biphasic Calcium Phosphate Bone Graft to BMP2 Improves New Bone Formation.

Bone morphogenetic protein-2 (BMP2) has been successfully utilized in dentistry to promote new bone formation because of its osteoinductive ability to recruit mesenchymal progenitor cells and induce their differentiation to bone-forming osteoblasts. Recently, novel biphasic calcium phosphate scaffolds have been developed with similar osteoinductive properties capable of forming ectopic bone formation. The aim of the present study was to assess whether the combination of BMP2 with this novel Biphasic Calcium Phosphate (BCP) scaffold may additionally promote new bone regeneration. Cylindrical bone defects measuring 2.5 mm were created bilaterally in the femurs of 18 Wistar rats. After 4 weeks, the following six groups were assessed for new bone formation by micro-computed tomography (CT) as well as histological assessment: 1) collagen scaffolds + 20 μg of BMP2; 2) collagen scaffolds + 50 μg of BMP2; 3) collagen scaffolds + 100 μg of BMP2; 4) BCP scaffolds + 20 μg of BMP2; 5) BCP scaffolds + 50 μg of BMP2; and 6) BCP scaffolds + 100 μg of BMP2. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining was utilized to assess osteoclast activity and osteoclast number. The release kinetics of BMP2 from both BCP and collagen scaffolds was investigated over a 14-day period. The results from present study demonstrate that BMP2 is able to promote new bone formation in a concentration dependant manner when loaded with either a collagen scaffolds or BCP scaffolds. Micro-CT analysis demonstrated significantly higher levels of new bone formation in groups containing BCP + BMP2 when compared with collagen scaffolds + BMP2. BMP2 had little effect on osteoclast activity; however, less TRAP staining and osteoclast number was observed in the defects receiving collagen scaffolds when compared with BCP scaffolds. The release of BMP2 over time was rapidly released after 1 day on BCP scaffolds whereas a gradually release over time was observed for collagen scaffolds up to 14 days. The osteoinductive properties of BMP2 may further be enhanced by its combination with a novel synthetically fabricated osteoinductive BCP scaffold. Future clinical testing is required to further assess these preliminary findings.

Poly(trimethylene carbonate) and biphasic calcium phosphate composites for orbital floor reconstruction: a feasibility study in sheep

In the treatment of orbital floor fractures, bone is ideally regenerated. The materials currently used for orbital floor reconstruction do not lead to the regeneration of bone. Our objective was to render polymeric materials based on poly(trimethylene carbonate) (PTMC) osteoinductive, and to evaluate their suitability for use in orbital floor reconstruction. For this purpose, osteoinductive biphasic calcium phosphate (BCP) particles were introduced into a polymeric PTMC matrix. Composite sheets containing 50 wt% BCP particles were prepared. Also laminates with poly(D,L-lactide) (PDLLA) were prepared by compression moulding PDLLA films onto the composite sheets. After sterilisation by gamma irradiation, the sheets were used to reconstruct surgically-created orbital floor defects in sheep. The bone inducing potential of the different implants was assessed upon intramuscular implantation. The performance of the implants in orbital floor reconstruction was assessed by cone beam computed tomography (CBCT). Histological evaluation revealed that in the orbital and intramuscular implantations of BCP containing specimens, bone formation could be seen after 3 and 9 months. Analysis of the CBCT scans showed that the composite PTMC sheets and the laminated composite sheets performed well in orbital floor reconstruction. It is concluded that PTMC/BCP composites and PTMC/BCP composites laminated with PDLLA have osteoinductive properties and seem suitable for use in orbital floor reconstruction.

Histological Evaluation of Degradable Guided Bone Regeneration Membranes Prepared from Poly(trimethylene carbonate) and Biphasic Calcium Phosphate Composites

SummaryIn oral and maxillofacial surgery, guided bone regeneration using barrier membranes is an important strategy to treat bone defects. The currently used barrier membranes have important disadvantages. Barrier membranes prepared from resorbable poly(trimethylene carbonate) (PTMC) performed as well as collagen barrier membranes. We hypothesized that composite membranes prepared from surface‐eroding PTMC and osteoinductive biphasic calcium phosphate (BCP) would enhance bone formation even further. Bicortical critical size defects in the mandibular angle of rats were covered on both sides with the membranes. After 2, 4, and 12 weeks the extent of bone formation in the defects and the soft tissue reaction towards the membranes was examined histologically. At 2 and 4 weeks, the formation of new bone was observed in defects covered with PTMC, PTMC‐BCP and Biogide collagen membranes. At 12 weeks, bone defects that were covered with PTMC membranes and control Biogide collagen membranes were fully filled with new formed bone. However, at this time point, defects covered with PTMC‐BCP composite membranes had not led to new bone in the defects. Instead a significant tissue reaction, likely to remaining BCP particles, was observed.

The osteoinductive potential of printable, cell‐laden hydrogel‐ceramic composites

Hydrogels used as injectables or in organ printing often lack the appropriate stimuli to direct osteogenic differentiation of embedded multipotent stromal cells (MSCs), resulting in limited bone formation in these matrices. Addition of calcium phosphate (CaP) particles to the printing mixture is hypothesized to overcome this drawback. In this study we have investigated the effect of CaP particles on the osteoinductive potential of cell-laden hydrogel-CaP composite matrices. To this end, apatitic nanoparticles have been included in Matrigel constructs where after the viability of embedded progenitor cells was assessed in vitro. In addition, the osteoinductive potential of cell-laden Matrigel containing apatitic nanoparticles was investigated in vivo and compared with composites containing osteoinductive biphasic calcium phosphate (BCP) microparticles after subcutaneous implantation in immunodeficient mice. Histological and immunohistochemical analysis of the tissue response as well as in vivo bone formation revealed that apatitic nanoparticles were osteoinductive and induced osteoclast activation, but without bone formation. The BCP particles were more effective in inducing elaborate bone formation at the ectopic location.

Influence of different polymeric gels on the ectopic bone forming ability of an osteoinductive biphasic calcium phosphate ceramic

To evaluate moldable osteoinductive putties for bone repair we combined microstructured biphasic calcium phosphate (BCP) particles with five different polymeric gels, carboxymethyl cellulose (CMC), Pluronic® F-127 (PLU), polyvinyl alcohol (PVA), chitosan (CHI) and alginate (ALG). In vitro gel dissolution showed that CMC, PLU and ALG gels dissolved rapidly (within hours), while the CHI gel took several days and the PVA gel did not dissolve within 2 weeks. Implanting the putty formulations into sheep muscle for 12 weeks demonstrated ectopic bone formation in the control BCP group as well as the putties prepared with dissolving gels (CMC, PLU, ALG and CHI). Bone was not seen in the putty comprising PVA. Quantitative data showed that the CMC and PLU gels did not significantly affect the osteoinductivity of BCP granules, while the ALG and CHI gels showed a significant decrease in bone formation. These results suggest that the dissolvability and chemistry of the gels may be factors affecting the osteoinduction of the putties.

Gene expressions of Collagen type I, ALP and BMP-4 in osteo-inductive BCP implants show similar pattern to that of natural healing bones

Osteo-inductive materials give rise to ectopic bone formation in vivo either in muscles or in subcutaneous tissue. However, the underlying molecular mechanism is totally unclear. To investigate the expression pattern of bone related genes in osteo-inductive materials, we performed quantitative PCR (qPCR) to detect the expressions of type I collagen, alkaline phosphatase (ALP) and bone morphogenetic protein 4 (BMP-4) in biphasic calcium phosphate (BCP) ceramics implanted in dorsal muscle of dogs. Bone formation in mandibular alveolus defects served as controls showing the expression patterns of these genes in natural healing bones. Histological examinations were performed to show the bone formation in osteo-inductive BCP implants. Data of qPCR indicated that all tested genes had a similar expression pattern with two peaks during the bone formation either in BCP implants or natural healing bones. Type I collagen and ALP were expressed at lower levels with delayed peak in BCP implant than that in natural healing bone. Higher BMP-4 expression level was detected in BCP ceramic implant than in natural healing bone at all the time points. These results demonstrated that expression patterns of bone-related genes in the inductive bone formation are similar to that of natural healing bone formation. As these three genes are important parameters for osteoblast activity in bone formation, our data provide clue to uncover the molecular mechanism of bone formation in osteo-inductive materials.