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.     

Bone regeneration using photocrosslinked hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles

Although rhBMP-2 has excellent ability to accelerate the repair of normal bone defects, limitations of its application exist in the high cost and potential side effects. This study aimed to develop a composite photopolymerisable hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (PH/rhBMP-2/NPs) as the bone substitute to realize segmental bone defect repair at a low growth factor dose. Firstly rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (rhBMP-2/NPs) were prepared and characterized by DLS and TEM. Composite materials, PH/rhBMP-2/NPs were developed and investigated by SEM-EDS as well as a series of physical characterizations. Using hMSCs as an in vitro cell model, composite photopolymerisable hydrogels incorporating NPs (PH/NPs) showed good cell viability, cell adhesion and time dependent cell ingrowth. In vitro release kinetics of rhBMP-2 showed a significantly lower initial burst release from the composite system compared with the growth factor-loaded particles alone or encapsulated directly within the hydrogel, followed by a slow release over time. The bioactivity of released rhBMP-2 was validated by alkaline phosphatase (ALP) activity as well as a mineralization assay. In in vivo studies, the PH/rhBMP-2/NPs induced ectopic bone formation in the mouse thigh. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by three dimensional micro-computed tomographic (μCT) imaging, histological analysis, and biomechanical measurements. Animals implanted with the composite hydrogel containing rhBMP-2-loaded nanoparticles underwent gradual resorption with more pronounced replacement by new bone and induced reunion of the bone marrow cavity at 12 weeks, compared with animals implanted with hydrogel encapsulated growth factors alone. These data provided strong evidence that the composite PH/rhBMP-2/NPs are a promising substitute for bone tissue engineering.     

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.     

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly ?-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 μg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing micro-computed tomography (μCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non-BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by μCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, micro-compression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects.     

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.     

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.     

Osteogenic evaluation of calcium/magnesium-doped mesoporous silica scaffold with incorporation of rhBMP-2 by synchrotron radiation-based μCT

The regenerative treatment of large osseous defects remains a formidable challenge in orthopedic surgery today. In the present study, we have synthesized biodegradable calcium/magnesium-doped silica-based scaffolds with hierarchically macro/mesoporous structure (CMMS), and incorporated recombinant human bone morphogenetic protein-2 (rhBMP-2) into the scaffolds to obtain a hybrid system for osteogenic factor delivery in the functional repair of bone defects. The developed CMMS/rhBMP-2 scaffolds presented interconnected porous network, macropores (200-500 μm) and mesopores (5.7 nm), as well as good bioactivity and biocompatibility and proper degradation rate. Combined with the capacity to deliver ions and growth factors, the CMMS/rhBMP-2 scaffolds significantly promoted the in vitro osteogenic differentiation of bone marrow stromal cells (bMSCs), as evidenced by the enhanced expression of Runx-2, osteopontin, osteocalcin and bone sialoprotein, and induced the ectopic bone formation in the thigh muscle pouches of mice. We further assessed the in vivo effects of CMMS/rhBMP-2 scaffolds in a rabbit femur cavity defect model by using synchrotron radiation-based μCT (SRμCT) imaging and histological analysis, indicating that the CMMS/rhBMP-2 scaffolds resulted in more bone regeneration compared to that observed with the CMMS scaffolds without rhBMP-2. Moreover, scaffolds with or without rhBMP-2 underwent gradual resorption and replacement with bone and almost disappeared at 12 weeks, while the dense CMMS/rhBMP-2 material showed slower degradation rate and promoted the least extensive neo-bone formation. This study suggested that the hybrid CMMS/rhBMP-2 scaffolds system demonstrates promise for bone regeneration in clinical case of large bone defects.     

The in vivo bone formation by mesenchymal stem cells in zein scaffolds

In our previous study, a three-dimensional zein porous scaffold was prepared. This scaffold showed proper mechanical properties, good biocompatibility, and controllable biodegradation. In addition, it allowed blood vessels to form inside in vivo. In the current study, we prepared the complexes of zein scaffolds and rabbit MSCs, and investigated ectopic bone formation in nude mice. Furthermore, we implanted them into the radius defects of rabbits and assessed whether they could be helpful in the repair of critical-sized bone defects. The results showed that the complexes of zein scaffolds and rabbit MSCs could undergo ectopic bone formation in the thigh muscle pouches of nude mice. More importantly, the complexes could lead to the repair of critical-sized radius defects in rabbits accompanied with blood vessels' formation, which clearly demonstrates promise for the treatment of bone defects through tissue engineering.     

Repair of long intercalated rib defects using porous beta-tricalcium phosphate cylinders containing recombinant human bone morphogenetic protein-2 in dogs

A new method to repair rib defects with biomaterials containing recombinant human bone morphogenetic protein-2 (rhBMP-2) is presented in this report. We had reported previously the successful regeneration of bony rib defects by placing a short chain of small beta-tricalcium phosphate (beta-TCP) cylinders on the intact periosteum. The multi-cylinder implants were ineffective in promoting rib repair when the periosteum was absent. By adding rhBMP-2 to the beta-TCP cylinders, we were able to promote rib bone regeneration in the presence or absence of the periosteum. The osteogenic capacity of the rhBMP-2/beta-TCP composite implant and the time required to complete regeneration were evaluated in a canine model. An 8cm long section of rib bone, including the periosteum, was removed and replaced with a chain of the rhBMP-2/beta-TCP cylinders. At 6 weeks after implantation, the ribs were restored to their original configuration and mechanical strength. The multi-cylinder beta-TCP implants were degraded and replaced by new bone in 12 weeks. This new degradable bone-inducing implant material has significant clinical potential for rib repair.     

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860 ± 110 nm), PLLA/HA (845 ± 140 nm) and PLLA/collagen/HA (310 ± 125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.     

Initial cell pre-cultivation can maximize ECM mineralization by human mesenchymal stem cells on silk fibroin scaffolds

Fast remineralization of bone defects by means of tissue engineering is one of many targets in orthopedic regeneration. This study investigated the influence of a range of pre-culture durations for human bone marrow derived mesenchymal stem cells (hMSC) before inducing differentiation into osteoblast-like cells. The aim was to find the conditions that lead to maximal extracellular matrix (ECM) mineralization, in terms of both amount and best distribution. Additionally, the influence of silk fibroin scaffold pore size on mineralization was assessed. The formation of mineralized ECM by hMSCs cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified for up to 72 days in culture using non-invasive time-lapse micro-computed tomography (micro-CT). ECM mineralization increased linearly 3 weeks after the beginning of the experiment with addition of differentiation medium. Biochemical end-point assays measured the amount of DNA, calcium deposits, alkaline phosphatase activity and cell metabolic activity to corroborate the hypothesis that an initial pre-culture period of hMSCs on silk fibroin scaffolds can accelerate mineralized ECM formation. According to the micro-CT analysis mineralization on silk fibroin scaffolds with pores of 112-224 μm diameter was most efficient with an initial cell pre-culture period of 9 days, showing 6.87±0.81× higher mineralization values during the whole cultivation period than without an initial cell pre-culture period.     

The role of muscle-derived stem cells in bone tissue engineering

The formation of bone and repair of bone defect requires a source of pluripotential mesenchymal stem cells. However, the capacity of the human body to generate bone components is limited. In this report, we show that the highly purified myogenic cells by the preplate technique have the capacity to differentiate into osteogenic lineage in vitro. The recombinant human bone morphogenic protein (rh-BMP-2) was immobilized on the molded gelatin composite. Primary muscle cells were isolated from newborn Wistar-rats calf muscle. The cells were then preplated in collagen-coated flasks. After six serial platings, the culture was enriched with small, round cells [pp6]. The effects of immobilized rhBMP-2 on the gelatin scaffold were evaluated by the analysis of alkaline phosphatase (ALP) and osteocalcin in culture medium after seedings of muscle-derived cells [pp6]. The results showed that the cells isolated from pp6 slow adhering cells possessed round mononuclear phenotype, marked ALP stain and matrix mineralization. The synthesis and secretion of ALP from pp6 muscle-derived cells were persistent higher than that of pp1-pp5 groups. The efficacy of rhBMP-2 immobilization on the gelatin scaffolds as manifested as the synthesis and secretion of ALP and osteocalcin from muscle-derived cells was always significantly higher than that of the control samples. In summary, our results suggest that the muscle-derived pp6 cells were capable of inducing and participating in bone formation. These results suggest that muscle tissue is a valuable resource for osteoprogenitor cells to be used in clinical practice to improve bone healing.     

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.     

Human urine-derived stem cells can be induced into osteogenic lineage by silicate bioceramics via activation of the Wnt/β-catenin signaling pathway

Human urine-derived stem cells (USCs) have great application potential for cytotherapy as they can be obtained by non-invasive and simple methods. Silicate bioceramics, including calcium silicate (CS), can stimulate osteogenic differentiation of stem cells. However, the effects of silicate bioceramics on osteogenic differentiation of USCs have not been reported. In this study, at first, we investigated the effects of CS ion extracts on proliferation and osteogenic differentiation of USCs, as well as the related mechanism. CS particles were incorporated into poly (lactic-co-glycolic acid) (PLGA) to obtain PLGA/CS composite scaffolds. USCs were then seeded onto these scaffolds, which were subsequently transplanted into nude mice to analyze the osteogenic differentiation of USCs and mineralization of extracellular matrix formed by USCs in vivo. The results showed that CS ion extracts significantly enhanced cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and expression of certain osteoblast-related genes and proteins. In addition, cardamonin, a Wnt/β-catenin signaling inhibitor, reduced the stimulatory effects of CS ion extracts on osteogenic differentiation of USCs, indicating that the observed osteogenic differentiation of USCs induced by CS ion extracts involves Wnt/β-catenin signaling pathway. Furthermore, histological analysis showed that PLGA/CS composite scaffolds significantly enhanced the osteogenic differentiation of USCs in vivo. Taken together, these results suggest the therapeutic potential of combining USCs and PLGA/CS scaffolds in bone tissue regeneration.     

Precipitation of nanohydroxyapatite on PLLA/PBLG/Collagen nanofibrous structures for the differentiation of adipose derived stem cells to osteogenic lineage

Tissue engineering and nanotechnology have enabled engineering of nanostructured materials to meet the current challenges in bone treatment owing to rising occurrence of bone diseases, accidental damages and defects. Poly(l-lactic acid)/Poly-benzyl-l-glutamate/Collagen (PLLA/PBLG/Col) scaffolds were fabricated by electrospinning and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). The abundance and accessibility of adipose derived stem cells (ADSC) may prove to be novel cell therapeutics for bone repair and regeneration. ADSCs were cultured on these scaffolds and were induced to undergo osteogenic differentiation in the presence of PBLG/n-HA for BTE. The cell-biomaterial interactions were analyzed using cell proliferation, SEM and CMFDA dye extraction techniques. Osteogenic differentiation of ADSC was confirmed using alkaline phosphatase activity (ALP), mineralization (ARS) and dual immunofluorescent staining using both ADSC marker protein and Osteocalcin, which is a bone specific protein. The utmost significance of this study is the bioactive PBLG/n-HA biomolecule introduced on the polymeric nanofibers to regulate and improve specific biological functions like adhesion, proliferation and differentiation of ADSC into osteogenic lineage. This was evident from the immunostaining and CMFDA images of ADSCs showing cuboidal morphology, characteristic of osteogenic lineage. The observed results proved that the PLLA/PBLG/Col/n-HA scaffolds promoted greater osteogenic differentiation of ADSC as evident from the enzyme activity and mineralization profiles for bone tissue engineering.     

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro.

Bone morphogenetic protein-2 (BMP-2) delivered in a suitable implantable matrix has the potential to repair local skeletal defects by inducing new bone formation from undifferentiated pluripotent stem cells resident in host tissue. In this study, we examined in vitro the potential of a derivatized hyaluronic acid (Hyaff-11) scaffold as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) in bone and cartilage repair therapies. Hyaff-11 scaffolds were fabricated using a phase inversion/particulate leaching method and soak-loaded with rhBMP-2. In vitro release kinetics of rhBMP-2, demonstrated using enzyme-linked immunosorbant assay and alkaline phosphatase (ALP) assay revealed a slow, sustained rhBMP-2 release during 28 days, with a cumulative release of 31.82% of the initial rhBMP-2 loaded. rhBMP-2 was released in bioactive form as demonstrated by ALP induction of pluripotent cell line, C3H10T1/2 (T1/2), down the osteoblast lineage when incubated with the release supernatants. rhBMP-2 retention in Hyaff-11 scaffolds was greater than that from collagen gels, which released most of the initially loaded rhBMP-2 by 14 days. rhBMP-2-loaded Hyaff-11 scaffolds were also seeded with T1/2 cells and evaluated at 3, 7, 14, and 28 days for viability and expression of osteoblast phenotype. Cells remained viable throughout the study and expressed a time- and dose-dependent ALP and osteocalcin expression in the rhBMP-2 groups. Based on these observations, Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBMP-2, release low levels of bioactive rhBMP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.     

In vitro mineralization and bone osteogenesis in poly(ε-caprolactone)/gelatin nanofibers

The implementation of bio-inspired strategies in developing scaffolds for the reconstruction of oral, craniofacial and bone skeletal tissues after injury or resection remains a challenge. Currently, advanced scaffolds comprising nanofibers endowed with biochemical/biophysical signaling capability offer great advantages in bone regeneration, because of their faithful mimesis of the characteristic size scales encountered in the fibrous network of the native extracellular matrix (ECM). In this study, we investigate the biological potential of nanofibers made of polycaprolactone and gelatin on guiding the regenerative mechanisms of bone. Contact angle measurements and environmental SEM investigations indicate a weak linkage of gelatin molecules to PCL chains, facilitating an efficient adhesion signal to cells up to 3 days of culture. In vitro studies performed on human mesenchymal stem cells (hMSC) until 3 weeks in culture medium with osteogenic supplementation, clearly showing the effectiveness of PCL/Gelatin electrospun scaffolds in promoting bone osteogenesis and mineralization. The increase of alkaline phosphatase activity (ALP) and gene expression of bone-related molecules (bone sialoprotein, osteopontin and osteocalcin), indicated by immunodetection and upregulation level of mRNA, confirm that proposed nanofibers promote the osteogenic differentiation of hMSC, preferentially in osteogenic medium. Moreover, the evidence of newly formed collagen fibers synthesis by SIRCOL and their mineralization evaluated by Alizarin Red staining and EDS mapping of the elements Ca, P and Mg corroborate the idea that native osteoid matrix is ultimately deposited. All these data suggest that PCL and gelatin electrospun nanofibers have great potential as osteogenesis promoting scaffolds for successful application in bone surgery. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3008-3019, 2012.     

A dual function of copper in designing regenerative implants

The supply of titanium implants which are widely used in orthopaedics with both regenerative and anti-microbial properties will achieve a great progress in bone regeneration. We asked, whether by appropriate concentrations of copper ions it will be possible both to inhibit growth of bacteria and stimulate biological responses in mesenchymal stem cells (MSC). Using titanium material which released galvanically deposited copper at concentrations from 0.3 to 1.75 mM, growth of planktonic Staphylococcus aureus was blocked and more importantly adherent bacteria were cleared from the material surface within 24 h. To test biological responses of human bone marrow derived MSC due to copper ions, we found that copper stimulated the proliferation of MSC in a narrow concentration range around 0.1 mM. Similar copper concentrations enhanced osteogenic differentiation of MSC when cells were cultured in osteogenic differentiation medium. We observed increased activity of alkaline phosphatase (ALP), higher expression of collagen I, osteoprotegerin, osteopontin and finally mineralization of the cells. We conclude that titanium implants that release copper ions can be effective against bacterial infections at higher concentrations of copper near the implant surface and can promote bone regeneration when its concentration becomes lower due to diffusion.