Bone Tissue Engineering Strategies Research Articles

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture

Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3–4cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to the loading direction. The ex vivo loading system established here can be further applied to study responses to mechanical loading in osteogenesis as well as callus maturation for better understanding of factors to consider in successful bone regeneration with mechanical factors.

Systematic comparison of biologically active foreign ions-codoped calcium phosphate microparticles on osteogenic differentiation in rat osteoporotic and normal mesenchymal stem cells.

Osteoporosis is a disease characterized by structural deterioration of bone tissue, leading to skeletal fragility with increased fracture risk. Calcium phosphates (CaPs) are widely used in bone tissue engineering strategies as they have similarities to bone apatite except for the absence of trace elements (TEs) in the CaPs. Bioactive glasses (BGs) have also been used successfully in clinic for craniomaxillofacial and dental applications during the last two decades due to their excellent potential for bonding with bone and inducing osteoblastic differentiation. In this study, we evaluated the osteogenic effects of the ionic dissolution products of the quaternary Si-Sr-Zn-Mg-codoped CaP (TEs-CaP) or 45S5 Bioglass® (45S5 BG), both as mixtures and separately, on rat bone marrow-derived mesenchymal stem cells (rOMSCs & rMSCs) from osteoporotic and normal animals, using an MTT test and Alizarin Red S staining. The materials enhanced cell proliferation and osteogenic differentiation, especially the combination of the BG and TEs-CaP. Analysis by quantitative PCR and ELISA indicated that the expression of osteogenic-specific genes and proteins were elevated. These investigations suggest that the TEs-CaP and 45S5 BG operate synergistically to create an extracellular environment that promotes proliferation and terminal osteogenic differentiation of both osteoporotic and normal rMSCs.

Biofabrication and Bone Tissue Regeneration: Cell Source, Approaches, and Challenges

The growing occurrence of bone disorders and the increase in aging population have resulted in the need for more effective therapies to meet this request. Bone tissue engineering strategies, by combining biomaterials, cells, and signaling factors, are seen as alternatives to conventional bone grafts for repairing or rebuilding bone defects. Indeed, skeletal tissue engineering has not yet achieved full translation into clinical practice because of several challenges. Bone biofabrication by additive manufacturing techniques may represent a possible solution, with its intrinsic capability for accuracy, reproducibility, and customization of scaffolds as well as cell and signaling molecule delivery. This review examines the existing research in bone biofabrication and the appropriate cells and factors selection for successful bone regeneration as well as limitations affecting these approaches. Challenges that need to be tackled with the highest priority are the obtainment of appropriate vascularized scaffolds with an accurate spatiotemporal biochemical and mechanical stimuli release, in order to improve osseointegration as well as osteogenesis.

Controlled Release of Vanadium from a Composite Scaffold Stimulates Mesenchymal Stem Cell Osteochondrogenesis.

Large bone defects often require the use of autograft, allograft, or synthetic bone graft augmentation; however, these treatments can result in delayed osseous integration. A tissue engineering strategy would be the use of a scaffold that could promote the normal fracture healing process of endochondral ossification, where an intermediate cartilage phase is later transformed to bone. This study investigated vanadyl acetylacetonate (VAC), an insulin mimetic, combined with a fibrous composite scaffold, consisting of polycaprolactone with nanoparticles of hydroxyapatite and beta-tricalcium phosphate, as a potential bone tissue engineering scaffold. The differentiation of human mesenchymal stem cells (MSCs) was evaluated on 0.05 and 0.025wt% VAC containing composite scaffolds (VAC composites) in vitro using three different induction media: osteogenic (OS), chondrogenic (CCM), and chondrogenic/osteogenic (C/O) media, which mimics endochondral ossification. The controlled release of VAC was achieved over 28days for the VAC composites, where approximately 30% of the VAC was released over this period. MSCs cultured on the VAC composites in C/O media had increased alkaline phosphatase activity, osteocalcin production, and collagen synthesis over the composite scaffold without VAC. In addition, gene expressions for chondrogenesis (Sox9) and hypertrophic markers (VEGF, MMP-13, and collagen X) were the highest on VAC composites. Almost a 1000-fold increase in VEGF gene expression and VEGF formation, as indicated by immunostaining, was achieved for cells cultured on VAC composites in C/O media, suggesting VAC will promote angiogenesis in vivo. These results demonstrate the potential of VAC composite scaffolds in supporting endochondral ossification as a bone tissue engineering strategy.

Amniotic fluid-derived stem cells mixed with platelet rich plasma for restoration of rat alveolar bone defect.

Stem cells isolated from the amniotic fluid have been shown as a promising candidate for cell therapy and tissue engineering. However, the experimental and preclinical applications of amniotic fluid-derived stem cells (AFSCs) in the very field of maxillofacial bone tissue engineering are still limited. In this study, rat AFSCs were successfully harvested and characterized in vitro. The rat AFSCs showed typical fibroblastoid morphology, stable proliferation activity and multi-differentiation potential. Flow-cytometry analysis demonstrated that these cells were positive for CD29, CD44, and CD90, while negative for hematopoietic markers such as CD34 and CD45. The regenerative performance of AFSCs-premixed with platelet rich plasma (PRP) gel in restoration of alveolar bone defect was further investigated using a modified rat maxillary alveolar defect model. Micro-computer tomography and histological examination showed a superior regenerative capacity of AFSCs-premixed with PRP gel at both 4 and 8 weeks after operation comparing with control groups. Moreover, the implanted AFSCs can survive in the defect site and directly participate in the bone tissue regeneration. Taken together, these results indicated the feasibility of an AFSCs-based alveolar bone tissue engineering strategy for alveolar defect restoration.

The proangiogenic potential of a novel calcium releasing composite biomaterial: Orthotopic in vivo evaluation.

In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

Cell aggregation enhances bone formation by human mesenchymal stromal cells.

The amount of bone generated using current tissue engineering approaches is insufficient for many clinical applications. Previous in vitro studies suggest that culturing cells as 3D aggregates can enhance their osteogenic potential, but the effect on bone formation in vivo is unknown. Here, we use agarose wells to generate uniformly sized mesenchymal stromal cell (MSC) aggregates. When combined with calcium phosphate ceramic particles and a gel prepared from human platelet-rich plasma, we generated a tissue engineered construct which significantly improved in vivo bone forming capacity as compared to the conventional system of using single cells seeded directly on the ceramic surface. Histology demonstrated the reproducibility of this system, which was tested using cells from four different donors. In vitro studies established that MSC aggregation results in an up-regulation of osteogenic transcripts. And finally, the in vivo performance of the constructs was significantly diminished when unaggregated cells were used, indicating that cell aggregation is a potent trigger of in vivo bone formation by MSCs. Cell aggregation could thus be used to improve bone tissue engineering strategies.

The Role of Three-Dimensional Scaffolds in Treating Long Bone Defects: Evidence from Preclinical and Clinical Literature-A Systematic Review.

Long bone defects represent a clinical challenge. Bone tissue engineering (BTE) has been developed to overcome problems associated with conventional methods. The aim of this study was to assess the BTE strategies available in preclinical and clinical settings and the current evidence supporting this approach. A systematic literature screening was performed on PubMed database, searching for both preclinical (only on large animals) and clinical studies. The following string was used: “(Scaffold OR Implant) AND (Long bone defect OR segmental bone defect OR large bone defect OR bone loss defect).” The search retrieved a total of 1573 articles: 51 preclinical and 4 clinical studies were included. The great amount of preclinical papers published over the past few years showed promising findings in terms of radiological and histological evidence. Unfortunately, this in vivo situation is not reflected by a corresponding clinical impact, with few published papers, highly heterogeneous and with small patient populations. Several aspects should be further investigated to translate positive preclinical findings into clinical protocols: the identification of the best biomaterial, with both biological and biomechanical suitable properties, and the selection of the best choice between cells, GFs, or their combination through standardized models to be validated by randomized trials.

Bone Tissue Engineering Under Xenogeneic-Free Conditions in a Large Animal Model as a Basis for Early Clinical Applicability.

For decades, researchers have been developing a range of promising strategies in bone tissue engineering with the aim of producing a significant clinical benefit over existing therapies. However, a major problem concerns the traditional use of xenogeneic substances for the expansion of cells, which complicates direct clinical transfer. The study's aim was to establish a totally autologous sheep model as a basis for further preclinical studies and future clinical application. Ovine mesenchymal stromal cells (MSC) were cultivated in different concentrations (0%, 2%, 5%, 10%, and 25%) of either autologous serum (AS) or fetal calf serum (FCS). With an increase of serum concentration, enhanced metabolic activity and proliferation could be observed. There were minor differences between MSC cultivated in AS or FCS, comparing gene and protein expression of osteogenic and stem cell markers, morphology, and osteogenic differentiation. MSC implanted subcutaneously in the sheep model, together with a nanostructured bone substitute, either in stable block or moldable putty form, induced similar vascularization and remodeling of the bone substitute irrespective of cultivation of MSC in AS or FCS and osteogenic differentiation. The bone substitute in block form together with MSC proved particularly advantageous in the induction of ectopic bone formation compared to the cell-free control and putty form. It could be demonstrated that AS is suitable for replacement of FCS for cultivation of ovine MSC for bone tissue engineering purposes. Substantial progress has been made in the development of a strictly xenogeneic-free preclinical animal model to bring future clinical application of bone tissue engineering strategies within reach.

Oxygen mapping: Probing a novel seeding strategy for bone tissue engineering.

ABSTRACTBone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen‐imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell‐seeding strategy. Immortalized hMSCs, pre‐cultured in a monolayer for 30–40% or 70–80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real‐time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle‐type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30–40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70–80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell‐seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two‐dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894–902. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Effect of BMPs and Wnt3a co-expression on the osteogenetic capacity of osteoblasts

In the present study, third‑generation autologous‑inactivated bone morphogenic protein2 (BMP2), BMP4, BMP6, BMP7, BMP9 and Wnt3a lentiviral vectors were constructed and integrated into the genome of MC3T3‑E1 murine mesenchymal stem cells (MMSCs) to produce osteoinductive factor gene‑modified MMSCs. The transfection efficiency of each osteoinductive factor was then determined by detecting the expression levels of runt related transcription factor2 (Runx2) mRNA. The cotransfection with combinations of two lentiviruses was performed, and the expression levels of bone γ‑carboxyglutamate protein and alkaline phosphatase in the MC3T3‑E1 cell culture supernatant were detected. The expression level of Runx2 mRNA was detected by reverse transcription‑polymerase chain reaction, and western blotting was performed to detect the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a. The results demonstrated that the recombinant lentiviruses were successfully transfected into MC3T3‑E1 cells. The relative expression levels of Runx2 mRNA were greatest in the BMP2 group, sequentially followed by the BMP4, BMP9, BMP7, Wnt3a and BMP6 groups. The results of cotransfection of MC3T3‑E1 cells (a total of 8groups) demonstrated that BMP‑2 and BMP‑7 exhibited the highest cotransfection efficiency. Western blot analysis demonstrated that following BMP2 and BMP7 cotransfection of MC3T3‑E1 cells, the protein expression levels of BMP2, BMP4, BMP6, BMP7, BMP9 and Wnt3a were increased compared with control cells. In conclusion, the third‑generation lentiviral vectors effectively improved the osteogenic efficiencies of MC3T3‑E1 cells, which provided an important theoretical basis and therapeutic strategy for bone reconstruction and tissue engineering.

Alveolar bone tissue engineering in critical-size defects of experimental animal models: a systematic review and meta-analysis.

Regeneration of large, 'critical-size' bone defects remains a clinical challenge. Bone tissue engineering (BTE) is emerging as a promising alternative to autogenous, allogeneic and biomaterial-based bone grafting. The objective of this systematic review was to answer the focused question: in animal models, do cell-based BTE strategies enhance regeneration in alveolar bone critical-size defects (CSDs), compared with grafting with only biomaterial scaffolds or autogenous bone? Following PRISMA guidelines, electronic databases were searched for controlled animal studies reporting maxillary or mandibular CSD and implantation of mesenchymal stem cells (MSCs) or osteoblasts (OBs) seeded on biomaterial scaffolds. A random effects meta-analysis was performed for the outcome histomorphometric new bone formation (%NBF). Thirty-six studies were included that reported on large- (monkeys, dogs, sheep, minipigs) and small-animal (rabbits, rats) models. On average, studies presented with an unclear-to-high risk of bias and short observation times. In most studies, MSCs or OBs were used in combination with alloplastic mineral-phase scaffolds. In five studies, cells were modified by ex vivo gene transfer of bone morphogenetic proteins (BMPs). The meta-analysis indicated statistically significant benefits in favour of: (1) cell-loaded vs. cell-free scaffolds [weighted mean difference (WMD) 15.59-49.15% and 8.60-13.85% NBF in large- and small-animal models, respectively]; and (2) BMP-gene-modified vs. unmodified cells (WMD 10.06-20.83% NBF in small-animal models). Results of cell-loaded scaffolds vs. autogenous bone were inconclusive. Overall, heterogeneity in the meta-analysis was high (I2 >90%). In summary, alveolar bone regeneration is enhanced by addition of osteogenic cells to biomaterial scaffolds. The direction and estimates of treatment effect are useful to predict therapeutic efficacy and guide future clinical trials of BTE. Copyright © 2016 John Wiley & Sons, Ltd.

Local delivery of iron chelators reduces in vivo remodeling of a calcium phosphate bone graft substitute

Iron chelators are known activators of the Hypoxia Includible Factor-1α (HIF-1α) pathway, a critical cellular pathway involved in angiogenic responses to hypoxia. Local delivery of these chelators has shown promise in bone tissue engineering strategies by inducing angiogenesis and osteogenesis. Hypoxic microenvironments are also a stimulus for osteoclast differentiation and resorptive activity, a process likely mediated by HIF-1α. In vitro, low doses of the iron chelator Deferoxamine (DFO) has shown to induce HIF-1α mediated osteoclast formation and function. However other studies have proposed an opposite in vitro effect likely through HIF independent mechanisms. To investigate use of these medications in bioceramic based bone tissue engineering strategies this study aimed to determine the in vivo effect of local delivery of iron chelators on bioceramic remodeling. A non-weight bearing cranial onlay model was used to assess monetite resorption and new bone formation in the presence or absence of a repeated delivery of two iron chelators, DFO and 1,10 Phenanthroline (PHT) at doses known to induce HIF. We found a marked reduction graft resorption and remodeling associated with iron chelation. This was correlated to a 3-fold reduction in osteoclast number at the bone graft interface. Iron is needed for mitochondrial biogenesis during osteoclastic differentiation and reducing extracellular iron levels may inhibit this process and possibly overpower any HIF induced osteoclast formation. Our findings suggest that these inexpensive and widely available molecules may be used to locally reduce bioceramic scaffold resorption and encourages future investigations of iron chelators as bone anti-resorptive agents in other clinical contexts. Statement of significanceLow doses of iron chelators can induce angiogenesis and osteogenesis in repairing bone by stimulating the oxygen sensitive gene; hypoxia inducible factor. These medications have potential to augment bioceramic based bone tissue engineering strategies without the downsides of protein-based growth factors. HIF activation is also known to stimulate osteoclast-mediated resorption and could potentially accelerate remodeling of biocermaics, however we have shown that the local delivery of iron chelation at doses known to induce HIF resulted in a reduction of monetite resorption and a significant decrease in osteoclast number at the bone graft interface. This maybe due to HIF independent mechanism. This is the first study to show a local effect of iron chelators in vivo on osteoclast-mediated resorption. This opens the potential of further study of these bifunctional medications to modulate resorption of biocermaics in environments where a prolonged presence of material is desired for graft site stability. Moreover these safe widely used medications can be explored to locally reduce osteoclasts in pathological bone resorption.

Strontium eluting nanofibers augment stem cell osteogenesis for bone tissue regeneration

Strontium is known to offer a therapeutic benefit to osteoporotic patients by promoting bone formation. Thus, toward engineering scaffolds for bone tissue regeneration we have prepared polymer nanocomposite scaffolds by electrospinning. Strontium carbonate nanoparticles (nSrCO3) were added to poly(ε-caprolactone) (PCL) at 10 and 20wt% to develop nanocomposite fibrous scaffolds (PCL/SrC10 and PCL/SrC20) with fiber diameter in the range of 300⿿500nm. Incorporation of nSrCO3 decreased crystallinity and the elastic modulus of PCL. The composite scaffolds released Sr2+ ions with up to 65ppm in 4days from the PCL/SrC20 scaffolds. Cell studies confirmed that the composite scaffold with 20% nSrCO3 enhanced proliferation of human mesenchymal stem cells in vitro. There was marked increase in mineral deposition up to four folds in PCL/SrC20 suggesting enhanced osteogenesis. This was corroborated by increased mRNA and protein expression of various osteogenic markers such as BMP-2, Osterix and Runx2 in the PCL/SrC20 fibers. Thus, incorporation of nSrCO3 in polymer scaffolds is a promising strategy for bone tissue engineering as an alternative to the use of labile growth factors to impart bioactivity to polymer scaffolds.

Bio-inspired cell membrane ingredient cholesterol-conjugated chitosan as a potential material for bone tissue repair

We prepared a cholesterol-conjugated chitosan(CHCS) material and evaluated its potential application as a bone tissue repair material by in vitro cell experiments. Cell proliferation, differentiation and morphology on CHCS membrane surfaces with different graft degrees were assessed in mouse pre-osteoblasts MC3T3-E1 cells. The results indicate that CHCS materials could promote the proliferation of MC3T3-E1 cells at low graft degrees, but the CHCS material with high graft degree inhibits the proliferation of cells in contrast to the pure chitosan membrane. However, the alkaline phosphatase(ALP) activity of MC3T3-E1 cells on different CHCS membrane surface increased with increasing graft degrees of cholesterol. The area of cells stretched onto the surface of CHCS materials was larger than on the surface of CS materials, and more microfilaments and stress fibers in cells were observed on CHCS materials than on the pure chitosan material surface. After 7 d, the expression of related osteogenic marker genes, such as runt-related transcription factor 2(Runx2), osterix(OSX), osteocalcin(OCN), osteopontin(OPN), ALP and collagen I(COL-I) were all up-regulated in CHCS materials to different degrees compared to pure chitosan material, which indicated that the CHCS materials facilitated MC3T3-E1 cell differentiation and maturation. Characterizing CHCS materials is useful in designing and developing strategies for bone tissue engineering.

Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches.

Molecular Mechanisms of PPAR-γ Governing MSC Osteogenic and Adipogenic Differentiation.

Sharing the same precursor cell lineage located in the bone marrow, mesenchymal stroma/stem cells (MSCs), osteoblasts and adipocytes have a reciprocal relationship in differentiation and function. The nuclear transcription factor peroxisome-proliferator- activated receptor-gamma (PPAR-γ) has been found expressed in both osteoblasts and adipocytes, as well as in MSCs, suggesting its crucial role in regulating adipocyte formation and osteoblast development. It has been observed in animal models that upregulated PPAR-γ activity results in bone loss where marrow adiposity is facilitated, while downregulated PPAR-γ activity leads to bone mass elevation. Evidence suggests that the dual function of PPAR-γ in either anti-osteoblastic or pro-adipocytic aspects is determined by its ligand. Furthermore, various cytokines and extracellular signaling pathways are involved in the transactivation of PPAR-γ, which can trigger the adipogenesis/osteoblastogenesis switch. PPAR-γ, therefore, shows tremendous potential in novel strategies for bone tissue engineering and clinical application. This review summarizes the regulatory function of PPAR-γ in MSC differentiation, as well as the cytokine and extracellular signaling pathways participating in the cross-talk between adipogenesis and osteoblastogenesis.

Establishment of a preclinical ovine screening model for the investigation of bone tissue engineering strategies in cancellous and cortical bone defects.

BackgroundNew tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone.MethodsRelated to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes.ResultsThe in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %.ConclusionsAs a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.

Mesenchymal Stem Cells Ageing: Targeting the "Purinome" to Promote Osteogenic Differentiation and Bone Repair.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852-1861, 2016. © 2016 Wiley Periodicals, Inc.

Synergistic Effects of Vascular Endothelial Growth Factor on Bone Morphogenetic Proteins Induced Bone Formation In Vivo: Influencing Factors and Future Research Directions

Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs), as key mediators in angiogenesis and osteogenesis, are used in a combined delivery manner as a novel strategy in bone tissue engineering. VEGF has the potential to enhance BMPs induced bone formation. Both gene delivery and material-based delivery systems were incorporated in previous studies to investigate the synergistic effects of VEGF and BMPs. However, their results were controversial due to variation of methods incorporated in different studies. Factors influencing the synergistic effects of VEGF on BMPs induced bone formation were identified and analyzed in this review to reduce confusion on this issue. The potential mechanisms and directions of future studies were also proposed here. Further investigating mechanisms of the synergistic effects and optimizing these influencing factors will help to generate more effective bone regeneration.