BMP‐2‐transduced human bone marrow stem cells enhance neo‐bone formation in a rat critical‐sized femur defect

Synthetic graft materials are considered as possible substitutes for cancellous bone, but lack osteogenic and osteoinductive properties. In this study, we investigated how composite scaffolds of βTCP containing osteogenic human bone marrow mesenchymal stem cells (hBMSCs) and osteoinductive bone morphogenetic protein‐2 (BMP‐2) influenced the process of fracture healing. hBMSCs were loaded into βTCP scaffolds 24 h before implantation in a rat critical‐sized bone defect. hBMSCs were either stimulated with rhBMP‐2 or transduced with BMP‐2 by gene transfer. The effect of both protein stimulation and gene transfer was compared for osteogenic outcome. X‐rays were conducted at weeks 0, 1, 3, 6, 9 and 12 post‐operatively. In addition, bone‐labelling fluorochromes were applied at 0, 3, 6 and 9 weeks. Histological analysis was performed for the amount of callus tissue and cartilage formation. At 6 weeks, the critical‐sized defect in 33% of the rats treated with the Ad‐BMP‐2‐transduced hBMSCs/βTCP scaffolds was radiographically bridged. In contrast, in only 10% of the rats treated with rhBMP2/hBMSCs, 12 weeks post‐treatment, the bone defect was closed in all treated rats of the Ad‐BMP‐2 group except for one. Histology showed significantly higher amounts of callus formation in both Ad‐BMP‐2‐ and rhBMP‐2‐treated rats. The amount of neocartilage was less pronounced in both BMP‐2‐related groups. In summary, scaffolds with BMP‐2‐transduced hBMSCs performed better than those with the rhBMP2/hBMSCs protein. These results suggest that combinations of osteoconductive biomaterials with genetically modified MSCs capable of secreting osteoinductive proteins may represent a promising alternative for bone regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Pooled thrombin‐activated platelet‐rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin‐activated, pooled, platelet‐rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9‐fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7‐fold increase) in constructs cultured with tPRP. Staining for human‐specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose‐derived cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Industrial approach in developing an advanced therapy product for bone repair

Mesenchymal stem cells (MSCs) are multipotent cells with therapeutic applications. The aim of our work was to develop an advanced therapy product for bone repair, associating autologous human adipose‐derived MSCs (ASCs) with human bone allograft (TBF; Phoenix^®). We drew up specifications that studied: (a) the influence of tissue collection procedures (elective liposuction or non‐invasive resection) and patient age on cell number and function; (b) monolayer cell culture conditions and osteodifferentiation and particularly the possibility of reducing stages of culture; and (c) the bone construct preparation and especially the comparison between two types of cells seeded on bone allograft (number of cultured processed lipoaspirate (PLA) cells and monolayer‐expanded ASCs) and cultured for 1, 2 and 3 weeks. The results showed that tissue harvesting techniques and patient age did not affect PLA cell number and ASC cloning efficiency. PLA cells can be directly osteodifferentiated (instead of culturing them in expansion medium first and then differentiating them) and these cells were able to mineralize when they were cultured in an osteogenic medium containing calcium chloride. PLA cells directly seeded on bone allograft for a minimum of 3 weeks of culture in this osteogenic medium expressed osteocalcin and colonized the matrix better than monolayer‐expanded ASCs. This work detailed the specifications of a pharmaceutical laboratory to develop an advanced therapy product and this current approach is promising for bone repair. Copyright © 2009 John Wiley & Sons, Ltd.     

Engineering axially vascularized bone in the sheep arteriovenous‐loop model

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)‐loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein‐2 (rhBMP‐2), were harvested and directly autotransplanted in combination with β‐tricalcium phosphate–hydroxyapatite (β‐TCP–HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP‐2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β‐TCP–HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro‐computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV‐loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP‐2 stimulation in about 12 weeks in the sheep AV‐loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies. Copyright © 2012 John Wiley & Sons, Ltd.     

Molecular‐ and microarray‐based analysis of diversity among resting and osteogenically induced porcine mesenchymal stromal cells of several tissue origin

Mesenchymal stromal cells (MSCs) play a pivotal role in modern therapeutic approaches in bone‐healing disorders. Although bone marrow‐derived MSCs are most frequently used, the knowledge that many other adult tissues represent promising sources for potent MSCs has gained acceptance. In the present study, the osteogenic differentiation potential of porcine skin fibroblasts (FBs), as well as bone marrow‐ (BMSCs), adipose tissue‐ (ASCs) and dental pulp‐derived stromal cells (DSCs) were evaluated. However, additional application of BMP‐2 significantly elevated the delayed osteogenic differentiation capacity of ASC and FB cultures, and in DSC cultures the supplementation of platelet‐rich plasma increased osteogenic differentiation potential to a comparable level of the good differentiable BMSCs. Furthermore, microarray gene expression performed in an exemplary manner for ASCs and BMSCs revealed that ASCs and BMSCs use different gene expression patterns for osteogenic differentiation under standard media conditions, as diverse MSCs are imprinted dependent from their tissue niche. However, after increasing the differentiation potential of ASCs to a comparable level as shown in BMSCs, a small subset of identical key molecules was used to differentiate in the osteogenic lineage. Until now, the importance of identified genes seems to be underestimated for osteogenic differentiation. Apparently, the regulation of transmembrane protein 229A, interleukin‐33 and the fibroblast growth factor receptor‐2 in the early phase of osteogenic differentiation is needed for optimum results. Based on these results, bone regeneration strategies of MSCs have to be adjusted, and in vivo studies on the osteogenic capacities of the different types of MCSs are warranted. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

Scaffold‐free cell sheet injection results in bone formation

We previously reported a new cell transplantation method in which mesenchymal stem cells (MSCs) were cultured as cell sheets. The cultured MSC sheets showed high alkaline phosphatase (ALP) activities and osteocalcin (OC) contents. In the present study, we transplanted such sheets by injection to assess whether the injectable MSC sheets could form bone tissue at subcutaneous sites. At 4 weeks after the subcutaneous injection, the injected areas showed hard mass formation. Each mass consisted of newly formed bone, as evaluated by radiographic, histological and gene expression analyses as well as three‐dimensional computed tomography (3D‐CT). Histological analyses revealed extracellular bone matrix together with osteocytes and active osteoblasts. Real‐time PCR analyses showed high ALP and OC mRNA expressions. We also injected the cell sheets into dead bone to determine whether the lost osteogenic potential could be rescued, and histological analyses revealed that the injected cell sheets supplied osteogenic potential to the dead bone. The present study clearly indicates that osteogenic MSC sheets can be transplanted via injection through a needle and that bone formation results in the injected areas. Owing to its usage of a needle for fabrication of in vivo bone tissue, this injection method can be applied as a minimally invasive approach for hard tissue reconstruction. Copyright © 2010 John Wiley & Sons, Ltd.     

Cultured cell‐derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells

Cell‐derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro‐environment. Therefore, cultured cell‐derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro‐environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell‐derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)‐derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.     

The effect of ex vivo dynamic loading on the osteogenic differentiation of genetically engineered mesenchymal stem cell model

Mechanical loading has been described as a highly important stimulus for improvements in the quality and strength of bone. It has also been shown that mechanical stimuli can induce the differentiation of mesenchymal stem cells (MSCs) along the osteogenic lineage. We have previously demonstrated the potent osteogenic effect of MSCs engineered to overexpress the BMP2 gene. In this study we investigated the effect of mechanical loading on BMP2‐expressing MSC‐like cells, using a special bioreactor designed to apply dynamic forces on cell‐seeded hydrogels. Cell viability, alkaline phosphatase (ALP) activity, BMP2 secretion and mineralized substance formation in the hydrogels were quantified. We found that cell metabolism increased as high as 6.8‐fold, ALP activity by 12.5‐fold, BMP2 secretion by 182‐fold and mineralized tissue formation by 1.72‐fold in hydrogels containing MSC‐like cells expressing BMP2, which were cultured in the presence of mechanical loading. We have shown that ex vivo mechanical loading had an additive effect on BMP2‐induced osteogenesis in genetically engineered MSC‐like cells. These data could be valuable for bone tissue‐engineering strategies of the future. Copyright © 2010 John Wiley & Sons, Ltd.     

A rapid method for obtaining mesenchymal stem cells and platelets from bone marrow aspirate

Mesenchymal stem cells (MSCs) and platelet‐rich plasma (PRP) are currently used alone or in combination for therapeutic applications especially for bone repair. We tested whether MSCs can be isolated from bone marrow (BM) aspirate using a commercially available kit commonly used to obtain PRP from peripheral blood (PB). Results revealed that mononuclear cells and platelets from both PB and BM could be efficiently isolated by obtaining a mononuclear and platelet rich fraction (PB‐MPRF and BM‐MPRF, respectively). Starting with comparable volumes, the number of platelets increased 1.5‐fold in BM‐MPRF compared to PB‐MPRF. The number of clonogenic cells in BM‐MPRF samples was significantly higher than whole BM samples as revealed by CFU‐F assay (54.92 ± 8.55 CFU‐F/1.5 x 10⁵ nucleated cells and 32.50 ± 12.43 CFU‐F/1.5 x 10⁵ nucleated cells, respectively). Cells isolated from BM‐MPRF after in vitro expansion fulfilled the definition of MSCs by phenotypic criteria, and differentiated along osteogenic, adipogenic and chondrogenic lineages following induction. Results showed that the kit isolated MSCs and platelets from BM aspirate. Isolated MSCs were further expanded in a laboratory and BM‐MPRF was used clinically following BM withdrawal for rapid intra‐operative cell therapy for the treatment of bone defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Short‐term evaluation of electromagnetic field pretreatment of adipose‐derived stem cells to improve bone healing

An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell‐based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose‐derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor β, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three‐dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short‐term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment. Copyright © 2012 John Wiley & Sons, Ltd.     

Mesenchymal stromal cells improve the osteogenic capabilities of mineralized agarose gels in a rat full‐thickness cranial defect model

The authors previously created HAp or CaCO₃ formed on or in agarose gels (HAp and CaCO₃ gels, respectively) as biocompatible and biodegradable bone graft materials. However, these gels have limitations for bone regeneration. Mesenchymal stromal cells (MSCs) have osteogenic potential and are considered useful for bone tissue engineering. The purpose of this study was to clarify the osteogenic abilities of MSCs loaded in HAp or CaCO₃ gels (MSC/HAp and MSC/CaCO₃ gels, respectively) using a rat cranial defect model compared to HAp and CaCO₃ gels alone. HAp, CaCO₃, MSC/Hap, and MSC/CaCO₃ gels were prepared for in vivo analyses and implanted into full‐thickness bone defects created in the rat cranium. All samples were assessed radiologically and histologically at 4 and 8 weeks after implantation. Using microfocus‐computed tomography, an increase in bone formation was observed in the MSC‐loaded gels compared to the gels alone. In addition, peripheral quantitative computed tomography revealed higher bone mineral contents in the MSC‐loaded gels compared to the gels alone. After transmission X‐ray diffraction analyses, the degree of apatite c‐axis orientation as a bone quality index of newly formed bone in the MSC‐loaded gels was close to that of living cranial bone. Histologically, more extensive bone formation was detected in the MSC‐loaded gels compared to gels alone. Overall, MSC/HAp and MSC/CaCO₃ gels showed equivalent efficacy for bone regeneration. These findings demonstrate that loading of MSCs into the gels strengthened their osteogenic ability and improved the quality of the newly formed bone. As a result, MSC‐loaded gels could represent viable therapeutic biomaterials for bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Co-culturing mesenchymal stem cells from bone marrow and periosteum enhances osteogenesis and neovascularization of tissue-engineered bone

Mesenchymal stem cells (MSCs) isolated from bone marrow and periosteum are often used as cellular sources for bone tissue engineering. This study showed that co‐cultured human bone marrow stem cells (hBMSCs) and periosteal‐derived stem cells (hPCs) resulted in a synergistic effect on osteogenic differentiation both in vitro and in vivo. Compared to hBMSCs and hPCs, co‐culturing MSCs showed abundant mineralization, robust calcium deposition, steadily increasing ALP activity, and upgraded mRNA expression of osteogenic specific genes (COL1A1, BMP‐2, osteopontin, osteocalcin) in vitro. Eight weeks after implantation of cellular β‐TCP scaffolds in immunodeficient mice, similar synergistic effects were confirmed during in vivo evaluation of total new bone formation, mature bone formation, and neovascularization. Based on these findings, the use of co‐cultured hBMSCs and hPCs can be recommended as a promising new approach for bone tissue engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Osteogenic potential of rat stromal cells derived from periodontal ligament

Various mesenchymal stromal cells (MSCs) have been applied to regenerative medicine. MSCs derived from periodontal tissue could also be a useful cell source for alveolar bone regeneration. However, only a few attempts of direct comparisons have been made between MSCs from periodontal tissues and those from other somatic tissues. The purpose of this study was to clarify the osteogenic characteristics of mesenchymal stromal cells derived from bone marrow (BMSCs), adipose tissue (ASCs) and periodontal ligament (PDLSCs). BMSCs, ASCs and PDLSCs were isolated from Fisher 344 rats. After 1 week of primary culture, stromal cells were subjected to cell surface analysis and osteogenic differentiation. The cells were subcultured for 2 weeks with and without osteogenic supplements (OS), followed by biochemical and histological analyses. With regard to cell surface antigens, all MSCs were positive for CD29 and CD90 and negative for CD45. With regard to osteogenic differentiation, BMSCs with OS had the highest ALP activity, calcium uptake and osteocalcin content. Without OS, PDLSCs had the highest levels of these bone differentiation markers. RT-PCR analysis and histological analysis showed similar trends. These results indicate that PDLSCs are an ideal candidate for alveolar bone regeneration.     

Evaluation of cross‐linked chitosan microparticles for bone regeneration

The objective of this preliminary study was to evaluate the applying of chitosan (CS)‐based microparticles (MPs) in bone regeneration in vivo. The CS MPs were fabricated using our scale‐up method, as previously described. Mesenchymal stem cells (MSC) were harvested from the femora and tibiae of Dark Agouti (DA) rats and seeded on CS MPs. An in vitro MSCs attachment experiment was conducted by trypsinizing the cells attached to the MPs at 5, 10, 20 and 30 h. Fluorescence images of MSCs attached to the MPs were taken at 24 and 48 h, using a LIVE/DEAD cell assay. The MSC/osteoblasts (OB) seeded on MPs were then cultured in vitro using osteogenic media and implanted into partial thickness bone defects in rat femurs. There were two groups of rats, including experimental animals and controls, for the in vivo studies. The experimental group were implanted with MSC‐seeded MPs and observed at 4 and 8 weeks. The control group of rats did not receive any implant material except the stainless steel plate to support the defect. Four rats per group were used for the study. The femurs were extracted at 4 and 8 weeks post‐implantation and bone formation at the defect site was analysed using radiography, microcomputed tomography (µCT) and histology. Among all groups, a significant increase in bone formation was observed in the experimental group at 8 weeks implantation. The results of this study suggested that CS MPs prove to be a successful biomaterial for bone regeneration. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model

Tissue‐engineered constructs combining bone marrow mesenchymal stem cells with biodegradable osteoconductive scaffolds are very promising for repairing large segmental bone defects. Synchronizing and controlling the balance between scaffold‐material resorption and new bone tissue formation are crucial aspects for the success of bone tissue engineering. The purpose of the present study was to determine, and compare, the osteogenic potential of ceramic scaffolds with different resorbability. Four clinically relevant granular biomaterial scaffolds (specifically, Porites coral, Acropora coral, beta‐tricalcium phosphate and banked bone) with or without autologous bone marrow stromal cells were implanted in the ectopic, subcutaneous‐pouch sheep model. Scaffold material resorption and new bone formation were assessed eight weeks after implantation. New bone formation was only detected when the biomaterial constructs tested contained MSCs. New bone formation was higher in the Porites coral and Acropora coral than in either the beta‐tricalcium phosphate or the banked bone constructs; furthermore, there was a direct correlation between scaffold resorption and bone formation. The results of the present study provide evidence that, among the biomaterials tested, coral scaffolds containing MSCs promoted the best new bone formation in the present study. Copyright © 2013 John Wiley & Sons, Ltd.     

Synergistic induction of early stage of bone formation by combination of recombinant human bone morphogenetic protein‐2 and epidermal growth factor

This study evaluates whether the combination of the rhBMP‐2 and various types of growth factors including EGF, FGF, PDGF and VEGF increases osteoinductivity compared to the single use of rhBMP‐2 through in vitro and in vivo study. Cultured human MSCs were treated with rhBMP‐2 only or in combination with growth factors. For in vivo evaluation, rhBMP‐2 only or with growth factors was implanted into the calvarial defect made on SD rats. Both EGF and PDGF significantly increased both ALP activity and expression level in hMSCs when treated in combination with rhBMP‐2 at 3 and 7 days of differentiation and significantly raised the accumulation of the calcium at day 14. Furthermore, micro‐CT scanning revealed that the EGF an FGF groups show significantly increased new bone surface ratio compared to the rhBMP‐2 only group and, the EGF treatment significantly up regulated percent bone volume and trabecular number at two weeks after the surgery. VEGF treatment also significantly raised trabecular number and FGF treatment significantly increased the trabecular thickness. Histological examination revealed that the EGF combination group showed enhanced bone regeneration than the rhBMP‐2 only group two weeks after the implantation. Even though the treatment of rhBMP‐2 with PDGF and FGF failed to show enhanced osteogenesis in vitro and in vivo simultaneously, these results suggest that the positive effect of the combination of EGF and rhBMP‐2 is expected to induce the bone formation earlier compared to the single use of rhBMP‐2 in vitro and in vivo. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.