The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

Biological matrices and bionanotechnology

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.     

Regenerating articular tissue by converging technologies

Scaffolds for osteochondral tissue engineering should provide mechanical stability, while offering specific signals for chondral and bone regeneration with a completely interconnected porous network for cell migration, attachment, and proliferation. Composites of polymers and ceramics are often considered to satisfy these requirements. As such methods largely rely on interfacial bonding between the ceramic and polymer phase, they may often compromise the use of the interface as an instrument to direct cell fate. Alternatively, here, we have designed hybrid 3D scaffolds using a novel concept based on biomaterial assembly, thereby omitting the drawbacks of interfacial bonding. Rapid prototyped ceramic particles were integrated into the pores of polymeric 3D fiber-deposited (3DF) matrices and infused with demineralized bone matrix (DBM) to obtain constructs that display the mechanical robustness of ceramics and the flexibility of polymers, mimicking bone tissue properties. Ostechondral scaffolds were then fabricated by directly depositing a 3DF structure optimized for cartilage regeneration adjacent to the bone scaffold. Stem cell seeded scaffolds regenerated both cartilage and bone in vivo.     

Enhanced Growth of Endothelial Precursor Cells on PCG-Matrix Facilitates Accelerated,Fibrosis-Free,Wound Healing: A Diabetic Mouse Model

Diabetes mellitus (DM)-induced endothelial progenitor cell (EPC) dysfunction causes impaired wound healing, which can be rescued by delivery of large numbers of ‘normal’ EPCs onto such wounds. The principal challenges herein are (a) the high number of EPCs required and (b) their sustained delivery onto the wounds. Most of the currently available scaffolds either serve as passive devices for cellular delivery or allow adherence and proliferation, but not both. This clearly indicates that matrices possessing both attributes are ‘the need of the day’ for efficient healing of diabetic wounds. Therefore, we developed a system that not only allows selective enrichment and expansion of EPCs, but also efficiently delivers them onto the wounds. Murine bone marrow-derived mononuclear cells (MNCs) were seeded onto a PolyCaprolactone-Gelatin (PCG) nano-fiber matrix that offers a combined advantage of strength, biocompatibility wettability; and cultured them in EGM2 to allow EPC growth. The efficacy of the PCG matrix in supporting the EPC growth and delivery was assessed by various in vitro parameters. Its efficacy in diabetic wound healing was assessed by a topical application of the PCG-EPCs onto diabetic wounds. The PCG matrix promoted a high-level attachment of EPCs and enhanced their growth, colony formation, and proliferation without compromising their viability as compared to Poly L-lactic acid (PLLA) and Vitronectin (VN), the matrix and non-matrix controls respectively. The PCG-matrix also allowed a sustained chemotactic migration of EPCs in vitro. The matrix-effected sustained delivery of EPCs onto the diabetic wounds resulted in an enhanced fibrosis-free wound healing as compared to the controls. Our data, thus, highlight the novel therapeutic potential of PCG-EPCs as a combined ‘growth and delivery system’ to achieve an accelerated fibrosis-free healing of dermal lesions, including diabetic wounds.     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.     

尼氟酸对晚期内皮祖细胞生物学特性的影响

目的:探讨ClCa通道抑制剂--尼氟酸(Niflumic,NFA)对大鼠骨髓来源的晚期内皮祖细胞(endothelial progenitor cells,EPCs)生物学特性的影响。方法:密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2完全培养液进行体外培养,以第三代或第四代的晚期EPCs作为靶细胞,应用RT-PCR检测晚期EPCs上是否存在Cl Ca通道标志基因TMEM16A和Cl Ca4的表达。采用CCK-8法、Ed U标记法、划痕实验、Boyden小室实验及Matrigel法分别检测10μmol/L NFA对细胞增殖、迁移及体外血管形成能力的影响;应用荧光定量PCR及流式细胞术检测内皮分化标志v WF和CD31基因及蛋白的表达。结果:晚期EPCs表达ClCa通道标志基因TMEM16A和ClCa4;NFA抑制晚期EPCs的迁移功能(P0.05);但对EPCs的增殖、分化及成血管能力有促进作用。NFA上调了晚期EPCs的CD31和v WF基因和蛋白表达。结论:NFA能促进EPCs的增殖、分化及成血管能力,抑制EPCs的迁移能力。NFA对EPCs生物学特性的这类影响将为心血管疾病治疗药物选择方面提供一定的参考依据。     

In vitro bone growth responds to local mechanical strain in three-dimensional polymer scaffolds

Mechanical stimulation plays a key role in healing and remodelling of bone tissue in vivo, and is used in bone tissue regeneration strategies in vitro. Although macroscopic compression of three-dimensional (3-D) seeded constructs can increase bone formation, it is not yet reported how this response is related to differences in local mechanical strains inside the scaffolds. In this study, we experimentally test the hypothesis that differences in local average of heterogeneous strains in a polymer scaffold will correlate with induced differences in the local biological response.Twenty-four poly(l-lactic acid) porous scaffolds seeded with rat bone cells were cultured first for 2 and 3 weeks under static conditions, respectively. Then for 1 week, half of the scaffolds were cyclically compressed (1.5%, 1 Hz), 1 h daily, with continuous perfusion (0.1 ml/min). The remaining half was kept under static conditions. The pore-surface strains in the scaffolds at the start of culture were calculated with micro-finite element modelling based on micro-Computed Tomography (μCT) images. The locations of mineralized nodules were determined from μCT images and coupled to the calculated strains.Detectable mineralized nodules (>10³ μm³) were only present in the loaded samples. Averages of absolute principal strains at the start of culture were significantly higher at nodule sites than at sites without a nodule.The results support the hypothesis that regenerating bone tissue in a 3-D porous scaffold responds to local mechanical strain. The methodology presented in this study can contribute design optimisation of tissue regeneration strategies relying on mechanical stimulation.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

CREB-binding protein silencing inhibits thrombin-induced endothelial progenitor cells angiogenesis

Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence from our group suggested that CREB-binding protein (CBP) plays an important role in thrombin-induced EPCs migration. However, whether CBP could regulate EPCs angiogenic properties is unknown. In the present study, we investigated whether CBP silencing could inhibit thrombin-induced EPCs angiogenesis. EPCs isolated from the bone marrow of Sprague–Dawley rats were cultured and identified, and then were treated by thrombin alone or combined with CBP-shRNA lentivirus. The effect of CBP silencing on EPCs proliferation was assessed using BrdU incorporation assay. Cell adhesion and tube formation were detected to evaluate the angiogenic functions. Finally, mRNA and protein expression of relevant angiogenic genes were examined by real-time PCR, western-blot, and enzyme-linked immunoassay respectively. Luciferase reporter gene assay was performed to evaluate NF-κB activity. Administration of thrombin significantly promoted EPCs proliferation and adhesion. Thrombin also increased the tube formation in Matrigel assay. However, these effects of thrombin were abolished by CBP gene silencing. CBP silencing also abrogated thrombin-induced increases of integrin β2 expression. In thrombin-induced EPCs, CBP silencing significantly decreased the secretion of VEGF, IL-6 and suppressed NF-κB activity. In conclusion, thrombin-induced EPCs proliferation, adhesion, and tube formation were inhibited by CBP silencing, indicating that CBP plays an important role in thrombin-induced EPCs neovascularization.     

In vitro Response of the Bone Marrow-Derived Mesenchymal Stem Cells Seeded in a Type-I Collagen-Glycosaminoglycan Scaffold for Skin Wound Repair Under the Mechanical Loading Condition

In order to achieve successful wound repair by regenerative tissue engineering using mesenchymal stem cells (MSCs), it is important to understand the response of stem cells in the scaffold matrix to mechanical stress.
To investigate the clinical effects of mechanical stress on the behavior of cells in scaffolds, bone marrow-derived mesenchymal stem cells (MSCs) were grown on a type-I collagen-glycosaminoglycan (GAG) scaffold matrix for one week under cyclic stretching loading conditions.
The porous collagen-GAG scaffold matrix for skin wound repair was prepared, the harvested canine MSCs were seeded on the scaffold, and cultured under three kinds of cyclic stretching loading conditions ( 0%: control, 5% strain, 15% strain ). After 7 days incubation, MSCs were evaluated histologically and immunohistochemically regarding the proliferation and differentiation.
Cultured MSCs in the high strain (15% strain) group showed activea-smooth muscle actin (α-SMA) expression and poor differentiation into type-I collagen-positive cells, whereas enhanced differentiation into type-I collagen positive cells and a lack ofa-SMA expression where shown in the lower stress (5% strain) group. These results suggest that mechanical stress may affect the proliferation and differentiation of stem cells, and subsequently the wound healing process, through attachment interactions between the stem cells and scaffold matrix. Our findings provide an additional consideration for clinical treatment of wound repair using regenerative tissue engineering.     

Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.     

Distribution of bone-marrow stromal cells in a 3D scaffold depending on the seeding method and the scaffold inside a surface modification

The distribution of bone-marrow stromal cells (BMSC) was studied in 3D polylactide scaffolds. Seeding of cells into the scaffold by the dynamic method (with the aid of a peristaltic pump) has been shown to provide distribution of cells throughout the entire scaffold volume, unlike the static method of seeding, in which the cell suspension is applied onto the scaffold surface. Unlike the cells seeded into the scaffold by the dynamic method, the cells seeded by the static method practically completely migrate from the scaffold on the dish for the first several days. It is revealed that BMSCs cultivated in 3D polylactide scaffolds modified by fibrin form colonies, whereas BMSCs cultivated inside scaffolds modified by collagen type 1 distribute all over the scaffold volume in the form of individual cells.     

Human Urine Derived Stem Cells in Combination with β-TCP Can Be Applied for Bone Regeneration

Bone tissue engineering requires highly proliferative stem cells that are easy to isolate. Human urine stem cells (USCs) are abundant and can be easily harvested without using an invasive procedure. In addition, in our previous studies, USCs have been proved to be able to differentiate into osteoblasts, chondrocytes, and adipocytes. Therefore, USCs may have great potential and advantages to be applied as a cell source for tissue engineering. However, there are no published studies that describe the interactions between USCs and biomaterials and applications of USCs for bone tissue engineering. Therefore, the objective of the present study was to evaluate the interactions between USCs with a typical bone tissue engineering scaffold, beta-Tricalcium Phosphate (β-TCP), and to determine whether the USCs seeded onto β-TCP scaffold can promote bone regeneration in a segmental femoral defect of rats. Primary USCs were isolated from urine and seeded on β-TCP scaffolds. Results showed that USCs remained viable and proliferated within β-TCP. The osteogenic differentiation of USCs within the scaffolds was demonstrated by increased alkaline phosphatase activity and calcium content. Furthermore, β-TCP with adherent USCs (USCs/β-TCP) were implanted in a 6-mm critical size femoral defect of rats for 12 weeks. Bone regeneration was determined using X-ray, micro-CT, and histologic analyses. Results further demonstrated that USCs in the scaffolds could enhance new bone formation, which spanned bone defects in 5 out of 11 rats while β-TCP scaffold alone induced modest bone formation. The current study indicated that the USCs can be used as a cell source for bone tissue engineering as they are compatible with bone tissue engineering scaffolds and can stimulate the regeneration of bone in a critical size bone defect.     

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

Proliferation of canine bone marrow derived mesenchymal stem cells on different nanomaterial based thin film scaffolds

Stem cell niche research uses nanotechnologies to mimic the extra-cellular microenvironment to promote proliferation and differentiation. The aim of designing different scaffolds is to simulate the best structural and environmental pattern for extracellular matrix. This experiment was designed to study the proliferative behaviour of canine bone marrow deriver mesenchymal stem cells (MSCs) on different nanomaterial based thin film scaffolds of carbon nanotubes (CNT), chitosan and poly ε-caprolactone. Similar number of cells was seeded on the scaffolds and standard cell culture flask, taken as control. Cells were maintained on DMEM media and relative number of metabolically active cells was determined by MTT assay up to day six of culture. Cells proliferated on control and all the scaffolds as the days progressed. Although proliferation rate was slow but no decline of cell number was noticed on the scaffolds during the study period. Initially, the cell proliferation was lower on CNT but as time progressed no significant difference was observed compared to control. The result indicated that nanomaterial based scaffolds reduce the proliferation rate of canine MSCs. However, canine MSCs adapted and proliferated better on CNT substrate in vitro and may be used as a scaffold component in canine tissue engineering in future.     

Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

The in vivo assessment of a novel scaffold containing heparan sulfate for tissue engineering with human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.