Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs)

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. Here, we investigate if BMP9 and Wnt/β-catenin act synergistically on odontogenic differentiation. Using the immortalized SCAPs (iSCAPs) isolated from mouse apical papilla tissue, we demonstrate that Wnt3A effectively induces early osteogenic marker alkaline phosphatase (ALP) in iSCAPs, which is reduced by β-catenin knockdown. While Wnt3A and BMP9 enhance each other's ability to induce ALP activity in iSCAPs, silencing β-catenin significantly diminishes BMP9-induced osteo/odontogenic differentiation. Furthermore, silencing β-catenin reduces BMP9-induced expression of osteocalcin and osteopontin and in vitro matrix mineralization of iSCAPs. In vivo stem cell implantation assay reveals that while BMP9-transduced iSCAPs induce robust ectopic bone formation, iSCAPs stimulated with both BMP9 and Wnt3A exhibit more mature and highly mineralized trabecular bone formation. However, knockdown of β-catenin in iSCAPs significantly diminishes BMP9 or BMP9/Wnt3A-induced ectopic bone formation in vivo. Thus, our results strongly suggest that β-catenin may play an important role in BMP9-induced osteo/ondontogenic signaling and that BMP9 and Wnt3A may act synergistically to induce osteo/odontoblastic differentiation of iSCAPs. It's conceivable that BMP9 and/or Wnt3A may be explored as efficacious biofactors for odontogenic regeneration and tooth engineering.     

Local suppression of pro-inflammatory cytokines and the effects in BMP-2-induced bone regeneration

The objective of this study is to investigate the effect of local inflammation suppression on the bone regeneration. Gelatin hydrogels incorporating mixed immunosuppressive triptolide-micelles and bone morphogenic protein-2 (BMP-2) were prepared. The controlled release of both the triptolide and BMP-2 from the hydrogels was observed under in vitro and in vivo conditions. When either J774.1 macrophage-like or MC3T3-E1 osteoblastic cells were cultured in the hydrogels incorporating mixed 2.5, 5 or 10 mg of triptolide-micelles and BMP-2, the expression level of pro- and anti-inflammatory cytokines including interleukin (IL)-6 and IL-10 was down-regulated, but the alkaline phosphatase (ALP) activity was promoted compared with those of hydrogels incorporating BMP-2 without triptolide-micelles. When implanted into a critical-sized bone defect of rats, the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 showed significantly lower number of neutrophils, lymphocytes, macrophages or dendritic and mast cells infiltrated into the defect, and lower expression level of IL-6, TNF-α, and IL-10 than those incorporating BMP-2 without triptolide-micelles. The reduced local inflammation responses at the defects implanted with the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 subsequently enhanced the bone regeneration thereat. It is concluded that the proper local modulation of inflammation responses is a promising way to achieve the enhanced bone regeneration.     

Bone regeneration around N-acetyl cysteine-loaded nanotube titanium dental implant in rat mandible

New strategies involving drugs loading onto implant surfaces are required to enhance osseointegration and shorten healing time after implantation. In this study, we examined the feasibility of N-acetyl cysteine (NAC)-loaded nanotube titanium (NLN-Ti) implants as a potential drug delivery system. To determine the effect of NLN-Ti in in vitro and in vivo, viability and ROS formation was assessed and enzyme-linked immunosorbant assay (ELISA), Western blot, micro-computed tomography (μ-CT), hematoxylin and eoxin (H&E) staining and immunohistochemical (IHC) analysis were done. In vitro, cell viability was increased and inflammatory responses and reduced oxidative stress-related defense were decreased with MC 3T3-E1 cells exposed to a sustained release of NAC from NLN-Ti implants. Following NLN-Ti implant installation, μ-CT revealed an increase of newly formed bone volume and bone mineral density in the mandibles of Sprague Dawley rats. Relatively well formed new bone was demonstrated in close contact to the NLN-Ti implant surface by H&E staining. IHC revealed significantly higher expression of bone morphogenetic protein-2, -7 and heme oxygenase-1, and reduced expression of receptor activator of nuclear factor-kappa B ligand. The data indicate that NLN-Ti implants enhance osseointegration and highlight the value of the small animal model in assessing diverse biological responses to dental implants.     

Nanoparticle delivery of stable miR-199a-5p agomir improves the osteogenesis of human mesenchymal stem cells via the HIF1a pathway

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.     

Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration

Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue's very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P < 0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration.     

The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor

Gene therapy using non-viral vectors that are safe and efficient in transfecting target cells is an effective approach to overcome the shortcomings of protein delivery of growth factors. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to deliver polyethylenimine (PEI)-plasmid DNA (pDNA) [encoding platelet derived growth factor-B (PDGF-B)] complexes. The PEI-pPDGF-B complexes were fabricated at amine (N) to phosphate (P) ratio of 10 and characterized for size, surface charge, and in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. The complexes were ∼100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. Confocal microscopy revealed significant proliferation of BMSCs on complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the complex-activated scaffolds following 4 weeks of implantation (14- and 44-fold higher) when compared to empty defects or empty scaffolds, respectively. Together, these findings suggest that non-viral PDGF-B gene-activated scaffolds are effective for bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.     

Controlling stem cell-mediated bone regeneration through tailored mechanical properties of collagen scaffolds

Mechanical properties of the extracellular matrix (ECM) play an essential role in cell fate determination. To study the role of mechanical properties of ECM in stem cell-mediated bone regeneration, we used a 3D in vivo ossicle model that recapitulates endochondral bone formation. Three-dimensional gelatin scaffolds with distinct stiffness were developed using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) mediated zero-length crosslinking. The mechanical strength of the scaffolds was significantly increased by EDC treatment, while the microstructure of the scaffold was preserved. Cell behavior on the scaffolds with different mechanical properties was evaluated in vitro and in vivo. EDC-treated scaffolds promoted early chondrogenic differentiation, while it promoted both chondrogenic and osteogenic differentiation at later time points. Both micro-computed tomography and histologic data demonstrated that EDC-treatment significantly increased trabecular bone formation by transplanted cells transduced with AdBMP. Moreover, significantly increased chondrogenesis was observed in the EDC-treated scaffolds. Based on both in vitro and in vivo data, we conclude that the high mechanical strength of 3D scaffolds promoted stem cell mediated bone regeneration by promoting endochondral ossification. These data suggest a new method for harnessing stem cells for bone regeneration in vivo by tailoring the mechanical properties of 3D scaffolds.     

Selective osteogenesis by a synthetic mineral inducing peptide for the treatment of osteoporosis

Mineralization in mammalian cells is accomplished by concerted regulation of protein-based extracellular matrix (ECM) components, such as non-collagenous proteins and collagen fibrils. In this study, we investigated the ability of a collagen-binding motif (CBM) peptide derived from osteopontin to selectively affect osteogenic or adipogenic differentiation in vitro and in vivo. In particular, increased osteogenic differentiation and decreased adipogenic differentiation were observed in human mesenchymal stem cells (hMSCs). Osteocalcin (OCN) protein expression in MC3T3-E1 cells without osteogenic inducers was then investigated following treatment with the CBM peptide. In ovariectomized (OVX) mice, estrogen deficiency induced osteoporosis and increased fat tissue deposition. However, after the CBM peptide or estradiol was injected into the OVX mice for 2 months, the increased serum OCN concentration and alkaline phosphate (ALP) activity were decreased in the estradiol-treated group (OVX-E) and the high-concentration CBM peptide-treated group (OVX-HP). Significant bone loss was also observed in the ovariectomized mice (OVX-PBS). In particular, the bone volume per total volume (BV/TV) and bone mineral density (BMD) were significantly decreased in the OVX mice; however, both of these markers were restored in the OVX-HP group, which also had significantly well-developed bone structure and bone formation. In contrast to the bone structural change, adipose tissue was increased in the OVX-PBS. However, a significant decrease in total fat and subcutaneous fat was observed in the low-concentration CBM peptide-treated group (OVX-LP) and the estradiol-treated group (OVX-E). Taken together, these results suggest that the CBM peptide could be an effective therapeutic agent for osteoporosis due to its selective stimulation of osteogenic differentiation, rather than adipogenesis.     

Enhancement of bone regeneration by dual release of a macrophage recruitment agent and platelet-rich plasma from gelatin hydrogels

Macrophages play an important role in regulating inflammatory responses and tissue regeneration. In the present study, their effect on bone remodeling is investigated by the simultaneous application of a macrophage recruiting agent, SEW2871 of a sphingosine-1 phosphate agonist, and platelet-rich plasma (PRP). The non-water soluble SEW2871 was solubilized in water through micelles formation with l-lactic acid grafted gelatin, and the resulting micelles with PRP were incorporated into gelatin hydrogels. Mixed SEW2871-micelles and PRP were released from gelatin hydrogels in a controlled fashion both in vitro and in vivo. In vitro migration assay revealed that the presence of PRP synergistically promoted SEW2871-induced macrophages migration. When applied to a bone defect of rats, the hydrogels incorporating mixed SEW2871-micelles and PRP recruited a higher number of macrophages than those hydrogels incorporating either SEW2871-micelles or PRP. The hydrogels incorporating mixed SEW2871-micelles and PRP enhanced the level of tumor necrosis factor (TNF)-α of pro-inflammatory cytokine, 3 days after application, while pro-inflammatory responses coupled with a significant increase in the expression level of osteoprotegerin (OPG) and interleukin (IL)-10 and transforming growth factor (TGF)-β₁ of anti-inflammatory cytokine were observed 10 days postoperatively. The hydrogels incorporating mixed SEW2871-micelles and PRP promoted bone regeneration to a significant great extent compared with those incorporating PBS and either SEW2871-micelles or PRP. It is concluded that macrophages recruitment contributed to PRP-induced bone regeneration.     

Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.     

Stimulation of bone growth following zinc incorporation into biomaterials

Rapid development of zinc biology has broadened the applications of Zn-incorporated biomaterials to tissue engineering but also raised concerns about the long-term safety of released Zn²⁺ ions. Clinical success hinges on the amount of incorporated zinc and subsequent optimized release sufficient to stimulate osseointegration. In this study, zinc is incorporated into the sub-surface of TiO₂ coatings by plasma immersion ion implantation and deposition (PIII&D). The Zn-implanted coatings show significant improvement compared to the “bulk-doped” coatings prepared by plasma electrolyte oxidation in terms of osteogenesis in vitro and in vivo. Molecular and cellular osteogenic activities demonstrate that rBMSCs cultured on the Zn-implanted coatings have higher ALP activity and up-regulated osteogenic-related genes (OCN, Col-I, ALP, Runx2) compared to the bulk-doped Zn coatings and controls. In vivo osseointegration studies conducted for 12 weeks on the rat model show early-stage new bone formation and the bone contact ratio (12 week) on the Zn-implanted coating is larger. The ZnT1 and ZIP1 gene expression studies demonstrate that the Zn-implanted coatings can better stimulate bone growth with reduced Zn release than those doped with zinc throughout the coatings.     

The effect of dose on rhBMP-2 signaling,delivered via collagen sponge,on osteoclast activation and in vivo bone resorption

While recombinant human bone morphogenetic protein (rhBMP)-2-based bone therapy presents potential osteoinductivity, it also leads concern due to transient osteoclast activation during early healing periods, ultimately limiting its clinical use. Therefore, we investigated in vivo and in vitro rhBMP-2 signaling which mediates early bone resorbing effect, depending on the dose, and attempted to inhibit this resorption phenomenon using NFAT inhibitor as a target molecule. High-dose of rhBMP-2 (20 μg/defect) enhanced osteoclast activation and the expression of bone resorption markers, compared to low dose (5 μg/defect) at one week after surgery in collagen sponge-delivered rat calvarial defect models. Interestingly, this trend was also observed in the expression of bone formation markers. In particular, rhBMP-2 upregulated RANKL expression, while it downregulated osteoprotegerin (OPG) expression, resulting in a dose-dependent increase in the ratio of RANKL to OPG. NFAT inhibitor (150 μm) treatment in vivo suppressed the high-dose effect of rhBMP-2 on both resorption and formation. In vitro results of rhBMP-2 signaling and NFAT inhibitor effects in rat mesenchymal stem cells showed similar trends as in vivo results. Microcomputer tomography-based evaluation at 4 weeks showed that combined treatment of NFAT inhibitor with 20 μg rhBMP-2 in vivo increased bone volume (BV) more than 20 μg rhBMP-2 alone, which showed little difference in BV compared to 5 μg of rhBMP-2. These results demonstrated that rhBMP-2 implantation concurrently signalized into enhanced osteoclastogenesis and osteoblastogenesis in vivo, dose-dependently. Ratio of RANKL/OPG might be an index for early bone resorbing activity of implanted rhBMP-2. A local cocktail treatment of NFAT inhibitor and high-dose rhBMP-2 might be an alternative to overcome early bone resorbing effects, thereby accelerating bone formation.     

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.     

A comparison of the performance of mono- and bi-component electrospun conduits in a rat sciatic model

Synthetic nerve conduits represent a promising strategy to enhance functional recovery in peripheral nerve injury repair. However, the efficiency of synthetic nerve conduits is often compromised by the lack of molecular factors to create an enriched microenvironment for nerve regeneration. Here, we investigate the in vivo response of mono (MC) and bi-component (BC) fibrous conduits obtained by processing via electrospinning poly(ε-caprolactone) (PCL) and gelatin solutions. In vitro studies demonstrate that the inclusion of gelatin leads to uniform electrospun fiber size and positively influences the response of Dorsal Root Ganglia (DRGs) neurons as confirmed by the preferential extensions of neurites from DRG bodies. This behavior can be attributed to gelatin as a bioactive cue for the cultured DRG and to the reduced fibers size. However, in vivo studies in rat sciatic nerve defect model show an opposite response: MC conduits stimulate superior nerve regeneration than gelatin containing PCL conduits as confirmed by electrophysiology, muscle weight and histology. The G-ratio, 0.71 ± 0.07 for MC and 0.66 ± 0.05 for autograft, is close to 0.6, the value measured in healthy nerves. In contrast, BC implants elicited a strong host response and infiltrating tissue occluded the conduits preventing the formation of myelinated axons.     

The influence of cellular source on periodontal regeneration using calcium phosphate coated polycaprolactone scaffold supported cell sheets

Cell-based therapy is considered a promising approach to achieving predictable periodontal regeneration. In this study, the regenerative potential of cell sheets derived from different parts of the periodontium (gingival connective tissue, alveolar bone and periodontal ligament) were investigated in an athymic rat periodontal defect model. Periodontal ligament (PDLC), alveolar bone (ABC) and gingival margin-derived cells (GMC) were obtained from human donors. The osteogenic potential of the primary cultures was demonstrated in vitro. Cell sheets supported by a calcium phosphate coated melt electrospun polycaprolactone (CaP-PCL) scaffold were transplanted to denuded root surfaces in surgically created periodontal defects, and allowed to heal for 1 and 4 weeks. The CaP-PCL scaffold alone was able to promote alveolar bone formation within the defect after 4 weeks. The addition of ABC and PDLC sheets resulted in significant periodontal attachment formation. The GMC sheets did not promote periodontal regeneration on the root surface and inhibited bone formation within the CaP-PCL scaffold. In conclusion, the combination of either PDLC or ABC sheets with a CaP-PCL scaffold could promote periodontal regeneration, but ABC sheets were not as effective as PDLC sheets in promoting new attachment formation.     

Successful matrix guided tissue regeneration of decellularized pulmonary heart valve allografts in elderly sheep

In vivo repopulation of decellularized allografts with recipient cells leads to a positive remodeling of the graft matrix in juvenile sheep. In light of the increasing number of heart valve replacements among older patients (>65 years), this study focused on the potential for matrix-guided tissue regeneration in elderly sheep. Pulmonary valve replacement was performed in seven-year old sheep using decellularized (DV), decellularized and CCN1-coated (RV), or decellularized and in vitro reendothelialized pulmonary allografts (REV) (n = 6, each group). CCN1 coating was applied to support re-endothelialization. In vitro re-endothelialization was conducted with endothelial-like cells derived from peripheral blood. Echocardiograms of all grafts showed adequate graft function after implantation and at explantation 3 or 6 months later. All explants were macroscopically free of thrombi at explantation, and revealed repopulation of the allografts on the adventitial side of valvular walls and proximal in the cusps. Engrafted cells expressed vimentin, sm α-actin, and myosin heavy chain 2, while luminal cell lining was positive for vWF and eNOS. Cellular repopulation of valvular matrix demonstrates the capacity for matrix-guided regeneration even in elderly sheep but is not improved by in vitro endothelialization, confirming the suitability of decellularized matrix for heart valve replacement in older individuals.     

RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200–500 μm and micropores of 2–5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.     

Improved implant osseointegration of a nanostructured titanium surface via mediation of macrophage polarization

The use of endosseous implanted materials is often limited by undesirable effects that may be due to macrophage-related inflammation. The purpose of this study was to fabricate a nanostructured surface on a titanium implant to regulate the macrophage inflammatory response and improve the performance of the implant. Anodization at 5 and 20 V as well as UV irradiation were used to generate hydrophilic, nanostructured TiO₂ surfaces (denoted as NT5 and NT20, respectively). Their surface characteristics and in vivo osseointegration as well as the inflammatory response they elicit were analyzed. In addition, the behavior of macrophages in vitro was evaluated. Although the in vitro osteogenic activity on the two surfaces was similar, the NT5 surface was associated with more bone formation, less inflammation, and a reduced CD68⁺ macrophage distribution in vivo compared to the NT20 and polished Ti surfaces. Consistently, further experiments revealed that the NT5 surface induced healing-associated M2 polarization in vitro and in vivo. By contrast, the NT20 surface promoted the pro-inflammatory M1 polarization, which could further impair bone regeneration. The results demonstrate the dominant role of macrophage-related inflammation in bone healing around implants and that surface nanotopography can be designed to have an immune-regulating effect in support of the success of implants.     

Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration

Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody-mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis.