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.     

Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2

Considerable research has been focused on the development of bone morphogenetic protein-2 (BMP-2) delivery system for homologous and efficient bone regeneration. The aim of the present study was to develop a collagen-based targeting bone repair system. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. We showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. In vitro functional study also demonstrated that collagen matrix could maintain higher bioactivity of BMP2-h than native BMP-2. When demineralized bone matrix (DBM) impregnated with BMP2-h was implanted subcutaneously in rats, homogeneous bone formation was observed. Moreover, in a rabbit mandible defect model, surgical implantation of collagen matrix loaded with BMP2-h exhibited remarkable osteoinductive properties and excellent homogeneous bone formation. Our studies suggested that this novel collagen-based BMP-2 targeting bone repair system induced better bone formation not only in quantity but also in quality. Similar approaches may also be used for the repair of other tissue injuries.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold

In this study, a heparin-conjugated poly(l-lactic-co-glycolic acid) (HP-PLGA) scaffold was developed for the sustained delivery of bone morphogenetic protein-2 (BMP-2), and then used to address the hypothesis that BMP-2 delivered from this scaffold could enhance ectopic bone formation. We found the amount of heparin conjugated to the PLGA scaffolds could be increased up to 3.2-fold by using scaffolds made from star-shaped PLGA, as compared to scaffolds made from linear PLGA, and that the release of BMP-2 from the HP-PLGA scaffold was sustained for at least 14 days in vitro. The BMP-2 released from the HP-PLGA scaffold stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 14 days in vitro, suggesting that the HP-PLGA scaffold delivery system releases BMP-2 in a bioactive form for a prolonged period. By contrast, BMP-2 release from unmodified (no heparin) PLGA scaffolds induced a transient increase in ALP activity for the first 3 days and a decrease thereafter. In vivo bone formation studies showed the BMP-2-loaded HP-PLGA scaffolds induced bone formation to a much greater extent than did either BMP-2-loaded unmodified PLGA scaffolds or unloaded (no BMP-2) HP-PLGA scaffolds, with 9-fold greater bone formation area and 4-fold greater calcium content in the BMP-2-loaded HP-PLGA scaffold group compared to the BMP-2-loaded unmodified PLGA scaffold group. Collectively, these results demonstrate that the HP-PLGA delivery system is capable of potentiating the osteogenic efficacy of BMP-2, and underscore its importance as a possible bone regeneration strategy.     

The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2

Bacterial cellulose (BC) is a nanofibrous biological material with attractive physicochemical properties and biocompatibility. Its fiber is similar to the collagenous fiber of bone. To explore if BC could be utilized as a localized delivery system to increase the local concentration of cytokines for tissue engineering, we prepared the BC scaffold from Acetobacter xylinum X-2 (A.?xylinum X-2) and investigated the osteogenic potential of the BC scaffold coated with bone morphogenetic protein-2 (BMP-2). The data showed that BC had a good biocompatibility and induced differentiation of mouse fibroblast-like C2C12 cells into osteoblasts in the presence of BMP-2 in?vitro, as demonstrated by alkaline phosphatase (ALP) activity assays. Within a certain range (0?～?3?μg/scaffold), the osteogenic activity of induced osteoblasts was positively correlated to the concentrations of BMP-2. In in?vivo subcutaneous implantation studies, BC scaffolds carrying BMP-2 showed more bone formation and higher calcium concentration than the BC scaffolds alone at 2 and 4 weeks, respectively. The ALP activity assay and the measurement of calcium concentration of BC scaffolds also showed that more new bone was developed in the BC scaffolds carrying BMP-2 than in the BC scaffolds alone. Our studies suggest that BC is a good localized delivery system for BMPs and would be a potential candidate in bone tissue engineering.     

In vitro evaluation of electrospun silk fibroin/nano-hydroxyapatite/BMP-2 scaffolds for bone regeneration

Bone tissue engineering by using osteoinductive scaffolds seeded with stem cells to promote bone extracellular matrix (ECM) production and remodeling has evolved into a promising approach for bone repair and regeneration. In order to mimic the ECM of bone tissue structurally and compositionally, nanofibrous silk fibroin (SF) scaffolds containing hydroxyapatite (HAP) nanoparticles and bone morphogenetic protein 2 (BMP-2) were fabricated in this study using electrospinning technique. The microstructure, mechanical property, biocompatibility, and osteogenic characteristics were examined. It was found that the HAP nanoparticles were successfully incorporated in the SF nanofibers (diameter, 200–500 nm). The mechanical properties of SF/HAP/BMP-2 composite scaffolds increased with HAP content when it was less than 20 wt%, after which the mechanical properties dropped as HAP content increased. Cell culture tests using bone marrow mesenchymal stem cells (BMSCs) showed that the scaffolds had good biocompatibility and promoted the osteogenic differentiation of BMSCs. Therefore, the electrospun SF/HAP/BMP-2 scaffolds may serve as a promising biomaterial for bone tissue engineering.     

BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.     

Apatite-coated collagen scaffold for bone morphogenetic protein-2 delivery

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. BMP-2 is clinically used for spine fusion and bone fracture healing. Commercially available BMP-2 uses a type I collagen scaffold as a carrier, but it only releases BMP-2 for a short period of time, which may release the bone formation efficacy. In the present study, we hypothesize that apatite coating of a collagen scaffold increases the release period as well as the osteogenic efficacy of BMP-2. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluids (SBFs). Apatite coating on collagen scaffolds was confirmed by X-ray diffraction, electron spectroscopy for chemical analysis, attenuated total reflectance-Fourier transform infrared spectroscopy, and scanning electron microscopy. The rate and period of BMP-2 release from apatite-coated collagen scaffolds varied depending on the concentration of SBFs used. The 5× and 10× SBF apatite-coated collagen scaffolds released 91.8%±11.5% and 82.2%±13.1% of their loaded BMP-2 over 13 days in vitro, respectively, whereas noncoated collagen scaffold released 98.3%±2.2% over the initial one day. BMP-2 released from apatite-coated collagen scaffold significantly increased the alkaline phosphatase activity of cultured osteoblasts, compared with BMP-2 released from noncoated collagen scaffold. Computed tomography and histomorphometry showed that BMP-2 delivery using apatite-coated collagen scaffolds resulted in 2.5-fold higher bone formation volume and 4.0-fold higher bone formation area than BMP-2 delivery using noncoated collagen scaffolds. This study shows that simple apatite coating of a collagen scaffold results in a BMP-2 carrier that renders long-term release of BMP-2 and dramatically enhances osteogenic efficacy.     

Effect of autologous bone marrow stromal cell seeding and bone morphogenetic protein-2 delivery on ectopic bone formation in a microsphere/poly(propylene fumarate) composite

A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 microg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2-loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2-impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 microg/mg BMP-2-loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2-impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.     

Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2

Segmental defect regeneration has been a clinical challenge. Current tissue-engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating bone regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (bone plate or external fixation). In this paper, we tested a scaffold that does not require a bone plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as bone morphogenetic protein-2 (BMP-2) carrier. Twenty-two scaffolds were implanted in 5mm segmental defects in rat femurs stabilized with K-wire for 6 and 15 weeks with and without 10 microg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro-computer tomography (microCT) imaging. The scaffolds in the BMP group maintained the bone length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the bone mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain bone length and allow successfully regeneration in segmental defects.     

Discontinuous release of bone morphogenetic protein-2 loaded within interconnected pores of honeycomb-like polycaprolactone scaffold promotes bone healing in a large bone defect of rabbit ulna

The choice of an appropriate carrier and its microarchitectural design is integral in directing bone ingrowth into the defect site and determining its subsequent rate of bone formation and remodeling. We have selected a three-dimensional polycaprolactone (PCL) scaffold with an interconnected honeycomb-like porous structure to provide a conduit for vasculature ingrowth as well as an osteoconductive pathway to guide recruited cells responding to a unique triphasic release of osteoinductive bone morphogenetic proteins (BMP) from these PCL scaffolds. We hypothesize that the use of recombinant human bone morphogenetic protein 2 (rhBMP2)-PCL constructs promotes rapid union and bone regeneration of a large defect. Results of our pilot study on a unilateral 15 mm mid-diaphyseal segmental rabbit ulna defect demonstrated enhanced bone healing with greater amount of bone formation and bridging under plain radiography and microcomputed tomography imaging when compared with an empty PCL and untreated group after 8 weeks postimplantation. Quantitative measurements showed significantly higher bone volume fraction and trabecular thickness, with lower trabecular separation in the rhBMP2-treated groups. Histology evaluation also revealed greater mature bone formation spanning across the entire scaffold region compared with other groups, which showed no bone regeneration within the central defect zone. We highlight that it is the uniqueness of the scaffold having a highly porous network of channels that promoted vascular integration and allowed for cellular infiltration, leading to a discontinuous triphasic BMP2 release profile that mimicked the release profile during natural repair mechanisms in vivo. This study serves as preclinical evidence demonstrating the potential of combining osteoinductive rhBMP2 with our PCL constructs for the repair of large defects in a large animal model.     

Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds

The ability to deliver, over time, biologically active osteogenic growth factors by means of designed scaffolds to sites of tissue regeneration offers tremendous therapeutic opportunities in a variety of musculoskeletal diseases. The aims of this study were to generate porous biodegradable scaffolds encapsulating an osteogenic protein, bone morphogenetic protein 2 (BMP-2), and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. BMP-2-encapsulated poly(DL-lactic acid) (PLA) scaffolds were generated by an innovative supercritical fluid process developed for solvent-sensitive and thermolabile growth factors. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on three-dimensional scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor-encapsulated scaffolds was observed after culture in an ex vivo model of bone formation developed on the basis of the chick chorioallantoic membrane model. BMP-2-encapsulated polymer scaffolds showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation and within diffusion chambers implanted into athymic mice as assessed by X-ray analysis and immunocytochemistry. The generation of three-dimensional biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculoskeletal tissue engineering.     

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model

We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Bone tissue engineering with novel rhBMP2-PLLA composite scaffolds

The aims of the present study were to fabricate a novel porous polylactic acid (PLLA) composite scaffold and evaluate the capacity of the scaffold in carrying recombinant bone morphogenetic protein 2 (rhBMP2) for engineering bone formation. The structures of the PLLA scaffolds were evaluated by SEM and the controlled release of rhBMP2 from the composite scaffolds was assayed by ELISA. Bone induction by the scaffolds loaded with or without rhBMP2 was performed in the calf muscle of twenty Wistar rats for 3, 7, 10, 14, and 28 days. Tissue specimens were examined by Masson's trichrome and von Kossa stainings, and immunohistochemistry of bone proteins. Our results indicated that a moderate foreign body reaction was found in control scaffolds, which lasted for 4 weeks. The addition of rhBMP2 to this novel scaffold dramatically alleviated the adverse responses to PLLA. Enhanced deposition of collagen matrix and endochondral formation were observed in rhBMP2-PLLA scaffolds at 7-10 days, compatible with an early release of rhBMP2 in the composite scaffolds. Bone sialoprotein and osteopontin were demonstrated simultaneously. Von Kossa staining was observed in the test group at 10-14 days. In conclusion, the PLLA scaffolds exhibited the capability of carrying rhBMP2 for inducing bone formation within 2 weeks. These results suggest that rhBMP2-PLLA scaffold may be applicable in tissue engineering.     

Comparative study of osteogenic potential of a composite scaffold incorporating either endogenous bone morphogenetic protein-2 or exogenous phytomolecule icaritin: an in vitro efficacy study

A local delivery system with sustained and efficient release of therapeutic agents from an appropriate carrier is desirable for orthopedic applications. Novel composite scaffolds made of poly (lactic-co-glycolic acid) with tricalcium phosphate (PLGA/TCP) were fabricated by an advanced low-temperature rapid prototyping technique, which incorporated either endogenous bone morphogenetic protein-2 (BMP-2) (PLGA/TCP/BMP-2) or phytomolecule icaritin (ICT) (PLGA/TCP/ICT) at low, middle and high doses. PLGA/TCP served as control. In vitro degradation, osteogenesis and release tests showed statistical differences among PLGA/TCP/ICT, PLGA/TCP and PLGA/TCP/BMP-2 groups, where PLGA/TCP/ICT had the desired slow release of bioactive icaritin in a dose-dependent manner, whereas there was almost no BMP-2 release from the PLGA/TCP/BMP-2 scaffolds. PLGA/TCP/ICT significantly increased more ALP activity, upregulated mRNA expression of osteogenic genes and enhanced calcium deposition and mineralization in rabbit bone marrow stem cells cultured on scaffolds compared with the other two groups. These results indicate the desired degradation rate, osteogenic capability and release property in PLGA/TCP/ICT composite scaffold, as icaritin preserved its bioactivity and structure after incorporation, while PLGA/TCP/BMP-2 did not show an initially expected osteogenic potential, owing to loss of the original bioactivity of BMP-2 during its incorporation and fabrication procedure. The results suggest that PLGA/TCP composite scaffolds incorporating osteogenic ICT might be a promising approach for bone tissue bioengineering and regeneration.     

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.     

壳聚糖-磷酸钙/骨形态发生蛋白2复合骨髓间充质干细胞促进兔脊柱的融合

背景：前期实验已经证明壳聚糖-磷酸钙/骨形态发生蛋白2复合材料能够促进兔脊柱融合。 目的：评价壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子支架材料在兔椎间融合中的应用效果。 方法：制备壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子支架材料,并与大鼠骨髓间充质干细胞在体外构成组织工程骨。摘除40只新西兰大白兔椎间盘,随机分为4组：空白对照组未植入任何材料,对照组植入自体髂骨,支架材料组植入壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子复合材料,实验组植入组织工程骨材料。 结果与结论：术后12周：①X射线片：对照组与实验组椎体融合,两组间融合节段生物力学强度大致相同,生物力学强度高于空白对照组与支架材料组(P < 0.05),且支架材料组高于空白对照组(P < 0.05)。②组织学切片：实验组与对照组有编织骨岛和新生毛细血管生成,支架材料组仅观察到壳聚糖支架网络,空白对照组未发现特殊组织结构。表明壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子复合鼠骨髓间充质干细胞能够明显促进脊柱融合。