Bone Tissue Engineering Constructs Research Articles

Biocompatibility and in vivo osteogenic capability of novel bone tissue engineering scaffold A-W-MGC/CS.

BackgroundThis study aims to investigate the biocompatibility and in vivo osteogenic capability of the novel bone tissue engineering scaffold apatite-wollastonite-magnetic glass ceramic/chitosan (A-W-MGC/CS).MethodsRabbit bone marrow stromal cells (BMSCs) were transfected with adenovirus-human bone morphogenetic protein-2-green fluorescent protein (Ad-hBMP2-GFP). The transfected BMSCs were then inoculated onto the scaffold material A-W-MGC/CS to construct tissue-engineered bone. The attachment and proliferation of BMSCs were observed by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) detection, respectively. Rabbit models of bone defects were established and divided into three groups. Experimental group 1 was implanted with prepared tissue-engineered bone. Experimental group 2 was implanted with A-W-MGC/CS without transfected BMSCs. The blank group was injected with transfected BMSCs, without implantation of any scaffold. In the 12th week after surgery, the repair of bone defect was observed by X-ray examination, and histological observations of the area of bone defect were performed.ResultsA-W-MGC/CS resulted in good BMSC attachment and had no obvious effects on cell proliferation. In experimental group 1, good repair of bone defect was observed, and the scaffold material degraded completely. In experimental group 2, new bone was formed, but its quality was poor. In the blank group, there was mainly filling of fibrous connective tissues with no observable bone defect repair.ConclusionA-W-MGC/CS possesses good biocompatibility and in vivo osteogenic capability for bone defect repair.

Spatial optimization in perfusion bioreactors improves bone tissue-engineered construct quality attributes.

Perfusion bioreactors have shown great promise for tissue engineering applications providing a homogeneous and consistent distribution of nutrients and flow-induced shear stresses throughout tissue-engineered constructs. However, non-uniform fluid-flow profiles found in the perfusion chamber entrance region have been shown to affect tissue-engineered construct quality characteristics during culture. In this study a whole perfusion and construct, three dimensional (3D) computational fluid dynamics approach was used in order to optimize a critical design parameter such as the location of the regular pore scaffolds within the perfusion bioreactor chamber. Computational studies were coupled to bioreactor experiments for a case-study flow rate. Two cases were compared in the first instance seeded scaffolds were positioned immediately after the perfusion chamber inlet while a second group was positioned at the computationally determined optimum distance were a steady state flow profile had been reached. Experimental data showed that scaffold location affected significantly cell content and neo-tissue distribution, as determined and quantified by contrast enhanced nanoCT, within the constructs both at 14 and 21 days of culture. However, gene expression level of osteopontin and osteocalcin was not affected by the scaffold location. This study demonstrates that the bioreactor chamber environment, incorporating a scaffold and its location within it, affects the flow patterns within the pores throughout the scaffold requiring therefore dedicated optimization that can lead to bone tissue engineered constructs with improved quality attributes.

Construction of tissue-engineered bone using a bioreactor and platelet-rich plasma.

The aim of the present study was to construct tissue-engineered bone using a bioreactor and platelet-rich plasma (PRP). Bone marrow mesenchymal stem cells (BMSCs) and β-tricalcium phosphate (β-TCP) were cultured in a perfusion bioreactor with PRP-containing medium for 21 days to form a BMSC-TCP composite. Rabbits were then implanted with the BMSC-TCP composite. The morphology of the implanted BMSC-TCP composite was observed three months after surgery by scanning electron microscopy and hematoxylin and eosin (H&E) staining. In addition, the expression of cluster of differentiation (CD)31 and von Willebrand factor (WF) in the implanted BMSC-TCP composite was detected using immunohistochemistry. Bone formation was determined by comprehensive testing Following culture in a perfusion bioreactor and PRP, the BMSCs adhered to the β-TCP scaffold and the secretion of extracellular matrix was observed. The spreading and proliferation of cells was found to be enhanced on the scaffold. Furthermore, the vascular endothelial cell markers CD31 and VEF, were positively expressed. Therefore, these results suggest that tissue-engineered bone may be constructed using a bioreactor and PRP. PRP, which contains multiple growth factors, may promote vascularization of tissue-engineered bone.

A composite demineralized bone matrix – Self assembling peptide scaffold for enhancing cell and growth factor activity in bone marrow

The need for suitable bone grafts is high; however, there are limitations to all current graft sources, such as limited availability, the invasive harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical problems, and degradation properties. The lack of osteoinductive properties is a common problem. As an allogenic bone graft, demineralized bone matrix (DBM) can overcome issues such as limited sources and comorbidities caused by invasive harvest; however, DBM is not sufficiently osteoinductive. Bone marrow has been known to magnify osteoinductive components for bone reconstruction because it contains osteogenic cells and factors. Mesenchymal stem cells (MSCs) derived from bone marrow are the gold standard for cell seeding in tissue-engineered biomaterials for bone repair, and these cells have demonstrated beneficial effects. However, the associated high cost and the complicated procedures limit the use of tissue-engineered bone constructs. To easily enrich more osteogenic cells and factors to DBM by selective cell retention technology, DBM is modified by a nanoscale self-assembling peptide (SAP) to form a composite DBM/SAP scaffold. By decreasing the pore size and increasing the charge interaction, DBM/SAP scaffolds possess a much higher enriching yield for osteogenic cells and factors compared with DBM alone scaffolds. At the same time, SAP can build a cellular microenvironment for cell adhesion, proliferation, and differentiation that promotes bone reconstruction. As a result, a suitable bone graft fabricated by DBM/SAP scaffolds and bone marrow represents a new strategy and product for bone transplantation in the clinic.

Prevascularization of biofunctional calcium phosphate cement for dental and craniofacial repairs

ObjectivesCalcium phosphate cement (CPC) is promising for dental and craniofacial repairs. Vascularization in bone tissue engineering constructs is currently a major challenge. The objectives of this study were to investigate the prevascularization of macroporous CPC via coculturing human umbilical vein endothelial cells (HUVEC) and human osteoblasts (HOB), and determine the effect of RGD in CPC on microcapillary formation for the first time. MethodsMacroporous CPC scaffold was prepared using CPC powder, chitosan liquid and gas-foaming porogen. Chitosan was grafted with Arg-Gly-Asp (RGD) to biofunctionalize the CPC. HUVEC and HOB were cocultured on macroporous CPC-RGD and CPC control without RGD for up to 42d. The osteogenic and angiogenic differentiation, bone matrix mineral synthesis, and formation of microcapillary-like structures were measured. ResultsRGD-grafting in CPC increased the gene expressions of osteogenic and angiogenic differentiation markers than those of CPC control without RGD. Cell-synthesized bone mineral content also increased on CPC-RGD, compared to CPC control (p<0.05). Immunostaining with endothelial marker showed that the amount of microcapillary-like structures on CPC scaffolds increased with time. At 42d, the cumulative vessel length for CPC-RGD scaffold was 1.69-fold that of CPC control. SEM examination confirmed the morphology of self-assembled microcapillary-like structures on CPC scaffolds. SignificanceHUVEC+HOB coculture on macroporous CPC scaffold successfully achieved prevascularization. RGD incorporation in CPC enhanced osteogenic differentiation, bone mineral synthesis, and microcapillary-like structure formation. The novel prevascularized CPC-RGD constructs are promising for dental, craniofacial and orthopedic applications.

Angiogenic/osteogenic response of BMMSCs on bone‐derived scaffold: Effect of hypoxia and role of PI3K/Akt‐mediated VEGF‐VEGFR pathway

Bone tissue deficiency is a common clinical challenge. Tissue-engineered bone constructs are an effective approach for the repair of orthopedic bone defects. Mimicking the essential components of the in vivo microenvironment is an efficient way to develop functional constructs. In this study, bone marrow-derived mesenchymal stromal cells (BMMSCs) were seeded into bone-derived scaffolds, a material with similar structure to natural bone. This was done under hypoxic conditions, an environment that imitates that experienced by BMMSCs in vivo. Our data indicate that hypoxia (5% O2 ) significantly increases the proliferation of BMMSCs seeded in scaffolds. As reflected by highly expressed osteogenesis- and angiogenesis-associated biomarkers, including vascular endothelial growth factor (VEGF), RUNX2, bone morphogenetic protein-2/4 and osteopontin, hypoxia also significantly increases the osteogenic and angiogenic responses of BMMSCs seeded into bone-derived scaffold composites. PI3K/Akt-mediated regulation of VEGF-activated VEGFR1/2 signaling is important for hypoxia-induced proliferative/osteogenic/angiogenic responses in BMMSC cellular scaffolds. The combination of bone-derived scaffolds and hypoxia is conducive to the differentiation of BMMSCs into functional tissue-engineered scaffold composites.

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Stem cell engineered bone with calcium-phosphate coated porous titanium scaffold or silicon hydroxyapatite granules for revision total joint arthroplasty

Aseptic loosening in total joint replacements (TJRs) is mainly caused by osteolysis which leads to a reduction of the bone stock necessary for implant fixation in revision TJRs. Our aim was to develop bone tissue-engineered constructs based on scaffolds of clinical relevance in revision TJRs to reconstitute the bone stock at revision operations by using a perfusion bioreactor system (PBRS). The hypothesis was that a PBRS will enhance mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation and will provide an even distribution of MSCs throughout the scaffolds when compared to static cultures. A PBRS was designed and implemented. Scaffolds, silicon substituted hydroxyapatite granules and calcium-phosphate coated porous TiAl6V4 cylinders, were seeded with MSCs and cultured either in static conditions or in the PBRS at 0.75 mL/min. Statistically significant increased cell proliferation and alkaline phosphatase activity was found in samples cultured in the PBRS. Histology revealed a more even cell distribution in the perfused constructs. SEM showed that cells arranged in sheets. Long cytoplasmic processes attached the cells to the scaffolds. We conclude that a novel tissue engineering approach to address the issue of poor bone stock at revision operations is feasible by using a PBRS.

A humanized tissue-engineered in vivo model to dissect interactions between human prostate cancer cells and human bone

Currently used xenograft models for prostate cancer bone metastasis lack the adequate tissue composition necessary to study the interactions between human prostate cancer cells and the human bone microenvironment. We introduce a tissue engineering approach to explore the interactions between human tumor cells and a humanized bone microenvironment. Scaffolds, seeded with human primary osteoblasts in conjunction with BMP7, were implanted into immunodeficient mice to form humanized tissue engineered bone constructs (hTEBCs) which consequently resulted in the generation of highly vascularized and viable humanized bone. At 12 weeks, PC3 and LNCaP cells were injected into the hTEBCs. Seven weeks later the mice were euthanized. Micro-CT, histology, TRAP, PTHrP and osteocalcin staining results reflected the different characteristics of the two cell lines regarding their phenotypic growth pattern within bone. Microvessel density, as assessed by vWF staining, showed that tumor vessel density was significantly higher in LNCaP injected hTEBC implants than in those injected with PC3 cells (p < 0.001). Interestingly, PC3 cells showed morphological features of epithelial and mesenchymal phenotypes suggesting a cellular plasticity within this microenvironment. Taken together, a highly reproducible humanized model was established which is successful in generating LNCaP and PC3 tumors within a complex humanized bone microenvironment. This model simulates the conditions seen clinically more closely than any other model described in the literature to date and hence represents a powerful experimental platform that can be used in future work to investigate specific biological questions relevant to bone metastasis.

A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone

ABSTRACTThe skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact ‘organ’ bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo.

Ectopic Osteogenesis of Allogeneic Bone Mesenchymal Stem Cells Loading on β-Tricalcium Phosphate in Canines

Bone mesenchymal stem cells are progenitor cells for mesenchymal tissues. The objective of this study was to evaluate the subcutaneous ectopic osteogenesis of allogeneic bone mesenchymal stem cells, which were loaded on β-tricalcium phosphate in canines without immunosuppressive therapy. Osteoinduced allogeneic bone mesenchymal stem cells were seeded onto a β-tricalcium phosphate scaffold to construct tissue-engineered bone. Four dogs (recipients) in the allogeneic group were subcutaneously implanted with the allogeneic bone mesenchymal stem cells/scaffold; four dogs (donors) in the autogeneic group were implanted with the autogeneic bone mesenchymal stem cells/scaffold complex; and four dogs in the control group were implanted with scaffold alone. Systemic immune responses were evaluated by measuring the T-lymphocyte CD4, CD8, and CD4/CD8 subsets of each group. Subcutaneous osteogenesis was compared between the three groups by histologic analysis at week 24 after implantation. Flow cytometry showed no significant differences in the number of CD4 and CD8 T cells and the CD4/CD8 T-cell ratios among the three groups. Histologically, at week 24, both the autogeneic and allogeneic complexes led to subcutaneous osteogenesis, whereas the control group alone did not. There were no significant differences in the percentage of osteogenic area between the allogeneic and the autogeneic complexes on histomorphometric analysis (p > 0.05), which was significantly higher than that produced by the control group alone (p < 0.001). The results demonstrate that osteoinduced, allogeneic bone mesenchymal stem cells loaded on β-tricalcium phosphate enhanced ectopic bone formation in canines without immunosuppressive therapy.

Construction of functional tissue-engineered bone using cell sheet technology in a canine model

The aim of the present study was to construct functional tissue-engineered bone with cell sheet technology and compare the efficacy of this method with that of traditional bone tissue engineering techniques. Canine bone mesenchymal stem cells (BMSCs) were isolated using density gradient centrifugation and then cultured. The BMSCs were induced to differentiate into osteoblasts and cultured in temperature-responsive culture dishes. The BMSCs detached automatically from the temperature-responsive culture dishes when the temperature was reduced to 20°C, forming an intact cell sheet. Demineralized bone matrix (DBM) and platelet-rich plasma (PRP) were prepared and used to construct a DBM/PRP/BMSC cell sheet/BMSC complex, which was implanted under the left latissimus dorsi muscle in a dog model. A DBM/PRP/BMSC complex was used as a control and implanted under the right latissimus dorsi muscle in the dog model. Immunoblot assays were performed to detect the levels of growth factors. Osteogenesis was observed to be induced significantly more effectively in the DBM/PRP/BMSC cell sheet/BMSC implants than in the DBM/PRP/BMSC implants. Immunoblot assay results indicated that the levels of the growth factors platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) in the experimental group were 3.2- and 2.5-fold higher compared with those in the control group, respectively. These results indicated that the BMSC cell sheets were functional and more effective than the control cell complex. Therefore, cell sheet technology may be used for the effective construction of functional tissue-engineered bone with ideal properties.

Design of Biomimetic and Bioactive Cold Plasma-Modified Nanostructured Scaffolds for Enhanced Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

The objective of this study was to design a biomimetic and bioactive tissue-engineered bone construct via a cold atmospheric plasma (CAP) treatment for directed osteogenic differentiation of human bone morrow mesenchymal stem cells (MSCs). Porous nanocrystalline hydroxyapatite/chitosan scaffolds were fabricated via a lyophilization procedure. The nanostructured bone scaffolds were then treated with CAP to create a more favorable surface for cell attachment, proliferation, and differentiation. The CAP-modified scaffolds were characterized via scanning electron microscope, Raman spectrometer, contact angle analyzer, and white light interferometer. In addition, optimal CAP treatment conditions were determined. Our in vitro study shows that MSC adhesion and infiltration were significantly enhanced on CAP modified scaffolds. More importantly, it was demonstrated that CAP-modified nanostructured bone constructs can greatly promote total protein, collagen synthesis, and calcium deposition after 3 weeks of culture, thus making them a promising implantable scaffold for bone regeneration. Moreover, the fibronectin and vitronection adsorption experiments by enzyme-linked immunosorbent assay demonstrated that more adhesion-mediated protein adsorption on the CAP-treated scaffolds. Since the initial specific protein absorption on scaffold surfaces can lead to further recruitment as well as activation of favorable cell functions, it is suggested that our enhanced stem cell growth and osteogenic function may be related to more protein adsorption resulting from surface roughness and wettability modification. The CAP modification method used in this study provides a quick one-step process for cell-favorable tissue-engineered scaffold architecture remodeling and surface property alteration.

Construction of tissue engineered bone modified by bone morphogenetic protein-2 and bone morphogenetic protein-7 genes

Objective To investigate the feasibility and advantages of constructing a novel tissue engineered bone through lentivirus transfection using β-tricalcium phosphate (β-TCP) and adipose derived stem cells (ADSCs) and modification with human bone morphogenetic protein 2 gene (BMP-2) and human bone morphogenetic protein 7 gene (BMP-7).Methods ADSCs derived from SD rats were passaged into the third generation before they were randomly divided into 4 groups:BMP-2 (Lv-BMP-2-transfected ADSCs),BMP-7 (Lv-BMP-7-transfected ADSCs),BMP-2+BMP-7 (ADSCs cotransfected by Lv-BMP-2 and Lv-BMP-7) and control (non-transfected ADSCs).The ADSCs at a concentration of 1 × 106/30 μL were seeded onto the β-TCP for compound culture in vitro.PicoGreen dsDNA assay evaluated the growth of ADSCs.The alkaline phosphatase (ALP) activity of ADSCs was measured.Alizarin red S staining and real-time PCR were used to test the osteoblast activities of ADSCs.Results After the ADSCs were seeded onto the scaffolds for 0 to 14 days,a similar trend was observed in the 4 groups regarding changes in the DNA amount.At 14 days after cell seeding,the BMP-2,BMP-7 and BMP-2 + BMP-7 groups had more calcific nodules than the control group,with the highest number in the BMP-2 + BMP-7 group.The ALP,mRNA and protein expressions of osteopontin and osteoealcin in the BMP-2 + BMP-7 group were significantly higher than those in the other 3 groups (P ＜ 0.05).X-ray at 6 weeks postoperation showed bone defects were almost healed in the BMP-2 + BMP-7 group but not in the other 3 groups.HE staining showed complete cortex bone and bone marrow in the BMP-2 + BMP-7 group but only a little amount of bone trabecula,cortex bone and bone marrow in the other 3 groups.Conclusions Tissue engineered bone can be constructed using β-TCP combined with ADSCs co-transfected with BMP-2 and BMP-7.As such tissue engineered bone has a great advantage of remarkably increased capability of osteogenesis,it can be used as a new means of treating bone defects. Key words: Bone morphogenetic proteins; Adipose tissue; Mesenchymal stem cells; Tissue engineering; Bone defects

Rat defect models for bone grafts and tissue engineered bone constructs

The development of optimized biomaterials for restoring bone defects has been ongoing for many years. Animal models for testing bone grafts and tissue engineered constructs are an important aspect of this research. New bone scaffolding systems need to be tested both in vitro and in vivo, but ultimately animal studies provide answers and confidence in their efficacy prior to clinical applications. A robust set of rat models for understanding bone development in response to implanted materials have been developed. This review will describe frequently used rat bone defect models, such as the calvarial defect, long bone defect, and maxillofacial bone defect models.

In VivoBone Regeneration Using Tubular Perfusion System Bioreactor Cultured Nanofibrous Scaffolds

The use of bioreactors for the in vitro culture of constructs for bone tissue engineering has become prevalent as these systems may improve the growth and differentiation of a cultured cell population. Here we utilize a tubular perfusion system (TPS) bioreactor for the in vitro culture of human mesenchymal stem cells (hMSCs) and implant the cultured constructs into rat femoral condyle defects. Using nanofibrous electrospun poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffolds, hMSCs were cultured for 10 days in vitro in the TPS bioreactor with cellular and acellular scaffolds cultured statically for 10 days as a control. After 3 and 6 weeks of in vivo culture, explants were removed and subjected to histomorphometric analysis. Results indicated more rapid bone regeneration in defects implanted with bioreactor cultured scaffolds with a new bone area of 1.23 ± 0.35 mm(2) at 21 days compared to 0.99 ± 0.43 mm(2) and 0.50 ± 0.29 mm(2) in defects implanted with statically cultured scaffolds and acellular scaffolds, respectively. At the 21 day timepoint, statistical differences (p<0.05) were only observed between defects implanted with cell containing scaffolds and the acellular control. After 42 days, however, defects implanted with TPS cultured scaffolds had the greatest new bone area with 1.72 ± 0.40 mm(2). Defects implanted with statically cultured and acellular scaffolds had a new bone area of 1.26 ± 0.43 mm(2) and 1.19 ± 0.33 mm(2), respectively. The increase in bone growth observed in defects implanted with TPS cultured scaffolds was statistically significant (p<0.05) when compared to both the static and acellular groups at this timepoint. This study demonstrates the efficacy of the TPS bioreactor to improve bone tissue regeneration and highlights the benefits of utilizing perfusion bioreactor systems to culture MSCs for bone tissue engineering.

Pre-vascularization of bone tissue-engineered constructs

Vascularization remains one of the primary obstacles in the repair of bone defects. In the previous issue of Stem Cell Research &Therapy, Pedersen and colleagues show that co-immobilization of endothelial cells and mesenchymal stem cells in a tissue-engineered construct can achieve functional microvascular networks in vivo. These very interesting findings, together with other state-of-the-art research in this field, are presented in this commentary. They highlight the vital role of mesenchymal stem cells as supporting cells to nascent blood vessels. The development of pre-vascularized implants by using clinically relevant cell sources, which could lead to rapid integration into the host tissue, would be of immense interest.

Near-Infrared Optical Imaging for Monitoring the Regeneration of Osteogenic Tissue-Engineered Constructs

Millions of cases of bone injury or loss due to trauma, osteoporosis, and cancer occur in the United States each year. Because bone is limited in its ability to regenerate, alternative therapy approaches are needed. Bone tissue engineering has the potential to correct musculoskeletal disorders through the development of cell-based substitutes for osteogenic tissue replacement. Multiple medical imaging techniques such as magnetic resonance microscopy (MRM) were investigated recently; these techniques are able to provide useful information on the anatomical and structural changes of developing bone. However, there is a need for noninvasive approaches to evaluate biochemical constituents and consequent compositional development associated with growing osteogenic constructs. In this study, near-infrared (NIR) optical imaging with a bone-specific NIR-targeted probe, IRDye® 800CW BoneTag™ (800CW BT), was applied in this study to longitudinally visualize regions of mineralization of tissue-engineered bone constructs in vivo. A fluorescent cell-based assay was performed to confirm the preferential binding of 800CW BT to the mineralized matrix of differentiated osteogenically driven human mesenchymal stem cells (hMSCs) in vitro. The hMSCs were seeded onto a biocompatible gelatin scaffold, allowed to develop, and implanted into a mouse model. Engineered constructs were examined in vivo using NIR imaging for bone mineralization, paired with MRM for verification of developing tissue. Results showed that NIR imaging with 800CW BT labeling can effectively assess the calcification of the developing osteogenic constructs, which is consistent with the analysis of excised tissue using NIR microscopy and histology. In conclusion, this study evaluated bone-like function of regenerating bone through tracking calcium deposition via NIR optical imaging with a fluorophore-labeled probe in a noninvasive manner.

Humanised xenograft models of bone metastasis revisited: novel insights into species-specific mechanisms of cancer cell osteotropism

The determinants and key mechanisms of cancer cell osteotropism have not been identified, mainly due to the lack of reproducible animal models representing the biological, genetic and clinical features seen in humans. An ideal model should be capable of recapitulating as many steps of the metastatic cascade as possible, thus facilitating the development of prognostic markers and novel therapeutic strategies. Most animal models of bone metastasis still have to be derived experimentally as most syngeneic and transgeneic approaches do not provide a robust skeletal phenotype and do not recapitulate the biological processes seen in humans. The xenotransplantation of human cancer cells or tumour tissue into immunocompromised murine hosts provides the possibility to simulate early and late stages of the human disease. Human bone or tissue-engineered human bone constructs can be implanted into the animal to recapitulate more subtle, species-specific aspects of the mutual interaction between human cancer cells and the human bone microenvironment. Moreover, the replication of the entire "organ" bone makes it possible to analyse the interaction between cancer cells and the haematopoietic niche and to confer at least a partial human immunity to the murine host. This process of humanisation is facilitated by novel immunocompromised mouse strains that allow a high engraftment rate of human cells or tissue. These humanised xenograft models provide an important research tool to study human biological processes of bone metastasis.