Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing

Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.     

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.     

Phytomolecule icaritin incorporated PLGA/TCP scaffold for steroid-associated osteonecrosis: Proof-of-concept for prevention of hip joint collapse in bipedal emus and mechanistic study in quadrupedal rabbits

Steroid-associated osteonecrosis (SAON) may lead to joint collapse and subsequent joint replacement. Poly lactic-co-glycolic acid/tricalcium phosphate (P/T) scaffold providing sustained release of icaritin (a metabolite of Epimedium-derived flavonoids) was investigated as a bone defect filler after surgical core-decompression (CD) to prevent femoral head collapse in a bipedal SAON animal model using emu (a large flightless bird). The underlying mechanism on SAON was evaluated using a well-established quadrupedal rabbit model. Fifteen emus were established with SAON, and CD was performed along the femoral neck for the efficacy study. In this CD bone defect, a P/T scaffold with icaritin (P/T/I group) or without icaritin (P/T group) was implanted while no scaffold implantation was used as a control. For the mechanistic study in rabbits, the effects of icaritin and composite scaffolds on bone mesenchymal stem cells (BMSCs) recruitment, osteogenesis, and anti-adipogenesis were evaluated. Our efficacy study showed that P/T/I group had the significantly lowest incidence of femoral head collapse, better preserved cartilage and mechanical properties supported by more new bone formation within the bone tunnel. For the mechanistic study, our in vitro tests suggested that icaritin enhanced the expression of osteogenesis related genes COL1α, osteocalcin, RUNX2, and BMP-2 while inhibited adipogenesis related genes C/EBP-ß, PPAR-γ, and aP2 of rabbit BMSCs. Both P/T and P/T/I scaffolds were demonstrated to recruit BMSCs both in vitro and in vivo but a higher expression of migration related gene VCAM1 was only found in P/T/I group in vitro. In conclusion, both efficacy and mechanistic studies show the potential of a bioactive composite porous P/T scaffold incorporating icaritin to enhance bone defect repair after surgical CD and prevent femoral head collapse in a bipedal SAON emu model.     

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Abstract

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.     

Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b

Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNAs (miRNAs) modulate the osteogenesis of osteoprogenitor cells, we aimed to engineer human adipose-derived stem cells (hASCs), a promising cell source for bone engineering, with miRNA-expressing baculovirus vectors. We constructed 4 baculoviruses each expressing 1 human miRNA (miR-26a, miR-29b, miR-148b, miR-196a) and verified that the miRNA-expressing baculovirus vectors augmented hASCs osteogenesis. Among these 4 miRNAs, miR-148b and miR-196a exerted more potent osteoinductive effects than miR-26a and miR-29b. Furthermore, we unveiled that co-transduction of hASCs with miR-148b-expressing and bone morphogenetic protein 2 (BMP-2)-expressing baculovirus vectors enhanced and prolonged BMP-2 expression, and synergistically promoted the in vitro osteogenic differentiation of hASCs. Implantation of the hASCs co-expressing BMP-2/miR-148b into critical-size (4 mm in diameter) calvarial bone defects in nude mice accelerated and potentiated the bone healing and remodeling, filling ≈94% of defect area and ≈89% of defect volume with native calvaria-like flat bone in 12 weeks, as judged from micro computed tomography, histology and immunohistochemical staining. Altogether, this study confirmed the feasibility of combining miRNA and growth factor expression for synergistic stimulation of in vitro osteogenesis and in vivo calvarial bone healing.     

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.     

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.     

Characterization of human ethmoid sinus mucosa derived mesenchymal stem cells (hESMSCs) and the application of hESMSCs cell sheets in bone regeneration

Mesenchymal stem cells (MSCs) have been extensively applied in the field of tissue regeneration. MSCs derived from various tissues exhibit different characteristics. In this study, a cluster of cells were isolated from human ethmoid sinus mucosa membrane and termed as hESMSCs. hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation. In particular, hESMSCs displayed strong osteogenic differentiation potential, and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Next, hESMSCs were prepared into a cell sheet and combined with a PSeD scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats, which showed excellent reparative effects. Additionally, ELISA assays revealed that secreted cytokines, such as BMP-2, BMP-4 and bFGF, were higher in the hESMSCs conditioned medium, and immunohistochemistry validated that hESMSCs cell sheet promoted the expression of BMP signaling downstream genes in newly formed bone. In conclusion, hESMSCs were demonstrated to be a class of mesenchymal stem cells that possessed high self-renewal capacity along with strong osteogenic potential, and the cell sheet of hESMSCs could remarkably promote new bone regeneration, indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone 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.     

Silicate bioceramics enhanced vascularization and osteogenesis through stimulating interactions between endothelia cells and bone marrow stromal cells

The facts that biomaterials affect the behavior of single type of cells have been widely accepted. However, the effects of biomaterials on cell–cell interactions have rarely been reported. Bone tissue engineering involves osteoblastic cells (OCs), endothelial cells (ECs) and the interactions between OCs and ECs. It has been reported that silicate biomaterials can stimulate osteogenic differentiation of OCs and vascularization of ECs. However, the effects of silicate biomaterials on the interactions between ECs and OCs during vascularization and osteogenesis have not been reported, which are critical for bone tissue regeneration in vivo. Therefore, this study aimed to investigate the effects of calcium silicate (CS) bioceramics on interactions between human umbilical vein endothelial cells (HUVECs) and human bone marrow stromal cells (HBMSCs) and on stimulation of vascularization and osteogenesis in vivo through combining co-cultures with CS containing scaffolds. Specifically, the effects of CS on the angiogenic growth factor VEGF, osteogenic growth factor BMP-2 and the cross-talks between VEGF and BMP-2 in the co-culture system were elucidated. Results showed that CS stimulated co-cultured HBMSCs (co-HBMSCs) to express VEGF and the VEGF activated its receptor KDR on co-cultured HUVECs (co-HUVECs), which was also up-regulated by CS. Then, BMP-2 and nitric oxide expression from the co-HUVECs were stimulated by CS and the former stimulated osteogenic differentiation of co-HBMSCs while the latter stimulated vascularization of co-HVUECs. Finally, the poly(lactic-co-glycolic acid)/CS composite scaffolds with the co-cultured HBMSCs and HUVECs significantly enhanced vascularization and osteogenic differentiation in vitro and in vivo, which indicates that it is a promising way to enhance bone regeneration by combining scaffolds containing silicate bioceramics and co-cultures of ECs and OCs.     

Effects of compatibility of deproteinized antler cancellous bone with various bioactive factors on their osteogenic potential

Combinations of calcium phosphate scaffolds and bioactive factors are promising niche-mimetic solutions for repairing large-sized bone defects. However, the importance of compatibility between scaffolds and bioactive factors on their osteogenic outcomes has been largely ignored. This study aimed to investigate the compatibility of calcinated antler cancellous bone (CACB) scaffolds with various bioactive factors including icariin (ICA), velvet antler polypeptides (VAP) or recombinant human bone morphogenetic protein-2 (rhBMP-2) as well as their combinational osteogenic potential in vitro and in vivo. Scanning electron microscopy and fourier transform infrared spectroscopy confirmed the uniform distribution and chemical stability of the reagents on CABC. In vitro release profiles showed relative steady release of ICA from ICA/CACB, burst VAP release from VAP/CACB, and minimal rhBMP-2 release from rhBMP-2/CACB composites. When compared with VAP and rhBMP-2, incorporation of ICA within CACB resulted in most increased cell attachment, proliferation, alkaline phosphatase activity, osteogenic gene expression, and mineralization of rat bone marrow mesenchymal stem cells. In rabbit mandible critical-sized defects, the most extensive osteogenesis and neovascularization were observed in the ICA/CACB group. Differences between the VAP/CACB and rhBMP-2/CACB groups were not apparent. Interestingly, low pro-inflammatory (TNF-α, IL-6) and high anti-inflammatory (IL-10) mRNA levels were observed at scaffold implantation sites which were in close association with amount of new bone formation. These findings highlight that the compatibility between scaffolds and bioactive factors should been taken into account when considering the formula of optimized bone defect repair.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.     

Radially oriented collagen scaffold with SDF-1 promotes osteochondral repair by facilitating cell homing

The migration of cells from the side and the bottom of the defect is important in osteochondral defect healing. Here, we designed a novel collagen scaffold that possessed channels in both the horizontal and the vertical directions, along with stromal cell-derived factor-1 (SDF-1) to enhance osteochondral regeneration by facilitating cell homing. Firstly we fabricated the radially oriented and random collagen scaffolds, then tested their properties. The radially oriented collagen scaffold had better mechanical properties than the random scaffold, but both supported cell proliferation well. Then we measured the migration of BMSCs in the scaffolds in vitro. The radially oriented collagen scaffold effectively promoted their migration, and this effect was further facilitated by addition of SDF-1. Moreover, we created osteochondral defects in rabbits, and implanted them with random or oriented collagen scaffolds with or without SDF-1, and evaluated cartilage and subchondral bone regeneration at 6 and 12 weeks after surgery. Cartilage regeneration with both the radially oriented scaffold and SDF-1 effectively promoted repair of the cartilage defect. Our results confirmed that the implantation of the radially oriented channel collagen scaffold with SDF-1 could be a promising strategy for osteochondral repair.     

Multifunctional and stable bone mimic proteinaceous matrix for bone tissue engineering

Biomaterial surface design with biomimetic proteins holds great promise for successful regeneration of tissues including bone. Here we report a novel proteinaceous hybrid matrix mimicking bone extracellular matrix that has multifunctional capacity to promote stem cell adhesion and osteogenesis with excellent stability. Osteocalcin-fibronectin fusion protein holding collagen binding domain was networked with fibrillar collagen, featuring bone extracellular matrix mimic, to provide multifunctional and structurally-stable biomatrices. The hybrid protein, integrated homogeneously with collagen fibrillar networks, preserved structural stability over a month. Biological efficacy of the hybrid matrix was proven onto tethered surface of biopolymer porous scaffolds. Mesenchymal stem cells quickly anchored to the hybrid matrix, forming focal adhesions, and substantially conformed to cytoskeletal extensions, benefited from the fibronectin adhesive domains. Cells achieved high proliferative capacity to reach confluence rapidly and switched to a mature and osteogenic phenotype more effectively, resulting in greater osteogenic matrix syntheses and mineralization, driven by the engineered osteocalcin. The hybrid biomimetic matrix significantly improved in vivo bone formation in calvarial defects over 6 weeks. Based on the series of stimulated biological responses in vitro and in vivo the novel hybrid proteinaceous composition will be potentially useful as stem cell interfacing matrices for osteogenesis and bone regeneration.     

Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects

Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.     

Enhanced bone tissue regeneration by antibacterial and osteoinductive silica-HACC-zein composite scaffolds loaded with rhBMP-2

Next-generation orthopedic implants with both osteoinductivity and antibacterial ability are greatly needed. In the present study, biodegradable rhBMP-2 loaded zein-based scaffolds with a macroporous structure were synthesized, and SBA-15 nanoparticles and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) were incorporated into the scaffolds to produce an anti-infective composite scaffold for delivery of osteogenic factors that facilitate the functional repair of bone defects. The silica/HACC/zein scaffolds developed here showed bioactivity, biocompatibility, and effective antibacterial activity. Confocal laser scanning microscopy (CLSM) was used to quantitatively measure the bactericidal efficacy with respect to bacterial adhesion. Results showed that the sample zein-HACC-S20 exhibited long-lasting antibacterial activity against Escherichia coli and Staphylococcus aureus up to 5 d. At a low dosage of rhBMP-2 (ca. 80 μg), the scaffolds released rhBMP-2 protein efficiently at a relatively slow rate, even after 27 d. An ALP activity and ECM mineralization assay showed that the zein-HACC-S20 scaffolds exhibited significant early osteogenic differentiation by generating enhanced ALP product on day 14 and ECM mineralization on day 21. In a mouse model of thigh muscle pouches, zein-S20 and zein-HACC-S20 groups resulted in obvious bone formation and gave more extensive mineralization to the implants than silica free groups, indicating effective bone induction in vivo. In a rabbit model of critical-sized radius bone defects (20 mm in length and 5 mm in diameter), the bone defects were almost fully repaired and bone marrow cavity recanalization was detectable by 3D micro-CT technique and histological analysis after 12 weeks. In this way, the zein-HACC-S20 scaffolds were proven to significantly promote the bone repair. They also demonstrated considerable promise for tissue engineering. Silica/HACC/zein scaffolds with both antibacterial activity and the ability to induce osteogenesis have immense potential in orthopedics and other biomedical applications.     

Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs

Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue-engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ-transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks after post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue-engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.     

The impact of PLGA scaffold orientation on in vitro cartilage regeneration

The success of in vitro cartilage regeneration provides a promising approach for cartilage repair. However, the currently engineered cartilage in vitro is unsatisfactory for clinical application due to non-homogeneous structure, inadequate thickness, and poor mechanical property. It has been widely reported that orientation of scaffolds can promote cell migration and thus probably contributes to improving tissue regeneration. This study explored the impact of microtubular oriented scaffold on in vitro cartilage regeneration. Porcine articular chondrocytes were seeded into microtubule-oriented PLGA scaffolds and non-oriented scaffolds respectively. A long-term in vitro culture followed by a long-term in vivo implantation was performed to evaluate the influence of scaffold orientation on cartilage regeneration. The current results showed that the oriented scaffolds could efficiently promote cell migration towards the inner region of the constructs. After 12 weeks of in vitro culture, the chondrocyte-scaffold constructs in the oriented group formed thicker cartilage with more homogeneous structure, stronger mechanical property, and higher cartilage matrix content compared to the non-oriented group. Furthermore, the in vitro engineered cartilage based on oriented scaffolds showed better cartilage formation in terms of size, wet weight, and homogeneity after 12-week in vivo implantation in nude mice. These results indicated that the longitudinal microtubular orientation of scaffolds can efficiently improve the structure and function of in vitro engineered cartilage.     

The effect of crosslinking heparin to demineralized bone matrix on mechanical strength and specific binding to human bone morphogenetic protein-2

Demineralized bone matrix (DBM) is a collagen-based scaffold, but its low mechanical strength and limited BMP-2 binding ability restrict its application in bone repair. It is known that heparin could be immobilized onto scaffolds to enhance their binding of growth factors with the heparin-binding domain. Here, we crosslinked heparin to DBM to increase its BMP-2 binding ability. To our surprise, the mechanical strength of DBM was also dramatically increased. The compression modulus of heparin crosslinked DBM (HC-DBM) have improved (seven-fold increased) under wet condition, which would allow the scaffolds to keep specific shapes in vivo. As expected, HC-DBM showed specific binding ability to BMP-2. Additional studies showed the bound BMP-2 exerted its function to induce cell differentiation on the scaffold. Subcutaneous implantation of HC-DBM carrying BMP-2 showed higher alkaline phosphatase (ALP) activity (2 weeks), more calcium deposition (4 and 8 weeks) and more bone formation than that of control groups. It is concluded that HC-DBM has increased mechanical intensity as well as specific BMP-2 binding ability; HC-DBM/BMP-2 enhances the osteogenesis and therefore could be an effective medical device for bone repair.