Repair of canine mandibular bone defects with bone marrow stromal cells and porous beta-tricalcium phosphate

Tissue engineering has become a new approach for repairing bone defects. Previous studies have been limited to the use of slow-degradable scaffolds with bone marrow stromal cells (BMSCs) in mandibular reconstruction. In this study, a 30 mm long mandibular segmental defect was repaired by engineered bone graft using osteogenically induced autologous BMSCs seeded on porous beta-tricalcium phosphate (beta-TCP, n=5). The repair of defects was compared with those treated with beta-TCP alone (n=6) or with autologous mandibular segment (n=4). In the BMSCs/beta-TCP group, new bone formation was observed from 4 weeks post-operation, and bony-union was achieved after 32 weeks, which was detected by radiographic and histological examination. In contrast, minimal bone formation with almost fibrous connection was observed in the group treated with beta-TCP alone. More importantly, the engineered bone with BMSCs/beta-TCP achieved a satisfactory biomechanical property in terms of bending load strength, bending displacement, bending stress and Young's modulus at 32 weeks post-operation, which was very close to those of contralateral edentulous mandible and autograft bone (p>0.05). Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and biodegradable beta-TCP can well repair the critical-sized segmental mandibular defects in canines.     

Repair of porcine articular osteochondral defects in non-weightbearing areas with autologous bone marrow stromal cells

In vivo niche is known to play important roles in terminal differentiation of implanted bone marrow stromal cells (BMSCs). This study explored the feasibility of repairing articular osteochondral defects using autologous BMSCs and biodegradable polymers. BMSCs from 18 hybrid pigs' marrows were either treated with dexamethasone (40 ng/mL) alone or chondrogenically induced with dexamethasone and transforming growth factor-beta1 (10 ng/mL). The cells were seeded respectively onto polylactic acid (PLA)- coated polyglycolic acid (PGA) scaffolds. Four osteochondral defects in each animal were created at non-weightbearing areas of knee joints (2/each side) and were respectively repaired by a chondrogenically induced BMSC-PGA/PLA construct in experimental group (Exp), by a dexamethasone-treated BMSC-PGA/PLA construct in control 1 group (Ctrl 1), by a PGA/PLA construct alone in control 2 group (Ctrl 2), or left unrepaired in control 3 group (Ctrl 3). To trace the implanted cells, green fluorescent protein (GFP)- labeled BMSCs were implanted in 2 animals. Gross view and histology showed that Exp and Ctrl 1 (with cell implantation) achieved better reparative results than Ctrl 2 and Ctrl 3 (without cell implantation) in terms of the reparative level and the restoration of the histological structure. In addition, 6-month results were better than 3-month results in all 4 groups. In Exp, 11 of 16 defects were completely repaired by hyaline cartilage and cancellous bone. In Ctrl 1, 11 of 16 defects were repaired by fibrocartilage and cancellous bone, although the repair with hyaline cartilage and cancellous bone was observed in 5 of 16 defects. In contrast, no obvious repair or only fibrotic tissue was observed in Ctrl 2 and Ctrl 3. The compressive moduli of repaired cartilage in Exp reached 80.27% of the normal amount at 6 months, with a high level of glycosaminoglycan (GAG) content (no statistical difference from normal). In Ctrl 1, the compressive moduli and GAG content were 62.69% and 78.03% of normal levels, respectively. More importantly, GFP-labeled cells were detected in the engineered cartilage and the repaired subchondral bone. These results strongly indicate that the implanted BMSCs can differentiate into either chondrocytes or osteoblasts and repair articular osteochondral defects by forming engineered cartilage and engineered bone.     

Tissue-engineered bone repair of goat-femur defects with osteogenically induced bone marrow stromal cells

Tissue engineering can generate bone tissue and has been shown to provide a better means of repairing weight-bearing bone defect. Previous studies, however, have heretofore been limited to the use of nonosteogenically induced bone marrow stromal cells (BMSCs) or the application of slow-degradation scaffolds. In this study, weight-bearing bone was engineered using osteogenically induced BMSCs. In addition, coral was used as a scaffold material, due to its proper degradation rate for the engineering and repair of a goat femur defect. A 25 mm long defect was created at the middle of the right femur in each of 10 goats. The rates of defect repair were compared in an experimental group of ten goats receiving implants containing osteogenically induced BMSCs and in the control group of goats (n = 10) receiving just coral cylinders. In the experimental group, bony union was observed by radiographic and gross view at 4 months, and engineered bone was further remodeled into newly formed cortexed bone at 8 months. There was increased gray density of radiographic rays in the repaired area, which was significantly different (p < 0.05) from that of the control group. H&E staining demonstrated that trabecular bone was formed at 4 months. Moreover, irregular osteon was observed at 8 months. Most importantly, the tissue-engineered bone segment revealed a similarity to the left-side normal femur in terms of bend load strength and bend rigidity, showing no significant difference (p > 0.05). In contrast, the coral cylinders of the control group showed no bone formation. Furthermore, almost complete resorption of the carrier had occurred, being evident at 2 months in the control group. H&E staining demonstrated that a small amount of residual coral particle was surrounded by fibrous tissue at 4 months whereas the residues disappeared at 8 months. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and coral can ideally heal critical-sized segmental bone defects in the weight-bearing area of goats.     

Osteogenic differentiation of human bone marrow stromal cells in hydroxyapatite-loaded microsphere-based scaffolds

Calcium-based minerals have consistently been shown to stimulate osteoblastic behavior in vitro and in vivo. Thus, use of such minerals in biomaterial applications has become an effective method to enhance bone tissue engineered constructs. In the present study, for the first time, human bone marrow stromal cells (hBMSC) were osteogenically differentiated on scaffolds consisting only of hydroxyapatite (HAp)-loaded poly(D,L-lactic acid-co-glycolic acid) (PLGA) microspheres of high monodispersity. Scaffold formulations included 0, 5, 10, and 20 wt% Hap, and the hBMSC were cultured for 6 weeks. Results demonstrated suppression of some osteogenic genes during differentiation in the HAp group, but higher end-point glycosaminoglycan and collagen content in 10% and 20% HAp samples, as evidenced by biochemical tests, histology, and immunohistochemistry. After 6 weeks of culture, constructs with 0% and 5% HAp had average compressive moduli of 0.7 ± 0.2 and 1.5 ± 0.9 kPa, respectively, whereas constructs with 10% and 20% HAp had higher average moduli of 17.6 ± 4.6 and 18.9 ± 8.1 kPa, respectively. The results of this study indicate that HAp inclusion in microsphere-based scaffolds could be implemented as a physical gradient in combination with bioactive signal gradients seen in previous iterations of these microsphere-based scaffolds to enhance osteoconduction and mechanical integrity of a healing site.     

The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds

We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSC attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD-modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK–PI3K–Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell–biomaterial interaction.     

Inverted colloidal crystal scaffolds with laminin-derived peptides for neuronal differentiation of bone marrow stromal cells

This study presents the effect of pore regularity on the preservation and differentiation of bone marrow stromal cells (BMSCs). Scaffolds with interconnected pores of inverted colloidal crystal (ICC) geometry were prepared by infiltrating chitosan-gelatin gels into the interstices of self-assembled microspheres, which were later dissolved with a solvent. In addition, the pore surfaces were grafted with two laminin-derived peptides (LDP). The experimental results revealed that the number of BMSCs in ICC scaffolds could increase 2.7-fold after cultivation over 7 days. Moreover, the distribution of cultured BMSCs in ICC scaffolds was quite uniform as compared with freeform scaffolds. ICC scaffolds could preserve 63% phenotypic BMSCs in average and freeform scaffolds 56%. The grafted LDP enhanced the adhesion efficiency of BMSCs in ICC scaffolds (about 70-75%) and produced NeuN-positive cells. A further induction with neuron growth factor could guide the differentiation of BMSCs toward mature neurons in LDP-grafted ICC scaffolds. The controlled topography of ICC structure and surface LDP can be promising in the cultivation of BMSCs and neural regeneration.     

Osteogenic differentiation of human bone marrow stromal cells on partially demineralized bone scaffolds in vitro

Tissue engineering has been used to enhance the utility of biomaterials for clinical bone repair by the incorporation of an osteogenic cell source into a scaffold followed by the in vitro promotion of osteogenic differentiation before host implantation. In this study, three-dimensional, partially demineralized bone scaffolds were investigated for their ability to support osteogenic differentiation of human bone marrow stromal cells (BMSCs) in vitro. Dynamic cell seeding resulted in homogeneous cell attachment and infiltration within the matrix and produced significantly higher seeding efficiencies when compared with a conventional static seeding method. Dynamically seeded scaffolds were cultured for 7 and 14 days in the presence of dexamethasone and evaluated on biochemical, molecular, and morphological levels for osteogenic differentiation. Significant elevation in alkaline phosphatase activity was observed versus controls over the 14-day culture, with a transient peak indicative of early mineralization on day 7. On the basis of RT-PCR, dexamethasone-treated samples showed elevations in alkaline phosphatase and osteocalcin expression levels at 7 and 14 days over nontreated controls, while bone sialoprotein was produced only in the presence of dexamethasone at 14 days. Scanning electron microscopy evaluation of dexamethasone-treated samples at 14 days revealed primarily cuboidal cells indicative of mature osteoblasts, in contrast to nontreated controls displaying a majority of cells with a fibroblastic cell morphology. These results demonstrate that partially demineralized bone can be successfully used with human BMSCs to support osteogenic differentiation in vitro. This osseous biomaterial may offer new potential benefits as a tool for clinical bone replacement.     

Differentiation of bone marrow stromal cells in poly(lactide-co-glycolide)/chitosan scaffolds

This study investigates the physicochemical properties of poly(lactide-co-glycolide) (PLGA)/chitosan scaffolds and the neuron growth factor (NGF)-guided differentiation of bone marrow stromal cells (BMSCs) in the scaffolds. The scaffolds were prepared by the crosslinking of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and genipin, and the differentiating BMSCs were characterized against CD44, CD90 and NeuN. The scaffold with 20% PLGA yielded 95% porosity, Young's modulus of 13 MPa, 70% adhesion of BMSCs and 1.6-fold increase in the cell viability over 7-day cultivation. BMSCs without guidance in the PLGA/chitosan scaffolds were prone to differentiate toward osteoblasts with apparent deposition of calcium. When NGF was introduced, an increased weight percentage of PLGA yielded more identified neurons. In addition, mature neurons emerged from the PLGA-rich biomaterials after induction with NGF over 2 days. A proper control over the physical and biomedical property of the scaffolds and the NGF-guided differentiation of BMSCs can be promising for nerve regeneration.     

可注射藻酸盐支架与人骨髓基质干细胞的体外复合培养*☆

背景：目前可注射组织工程骨的研究主要限于动物实验,若人骨髓基质干细胞与藻酸盐生物相容性良好,可注射组织工程骨将是极具前途的临床治疗手段。 目的：体外观察人骨髓基质干细胞与可注射支架藻酸钙凝胶的生物相容性。 方法：实验组将第2代人骨髓基质干细胞与藻酸钙凝胶复合培养,对照组单纯接种骨髓基质干细胞。倒置相差显微镜、扫描电镜观察各组细胞形态及增殖情况,MTT法半定量检测细胞增殖情况。 结果与结论：倒置显微镜下见实验组细胞生长良好,与对照组无明显差异。扫描电镜见骨髓基质干细胞在藻酸钙表面贴附、增殖良好,第6天时细胞已跨越微孔表面或向孔内生长。MTT法显示与对照组相比,实验组细胞增殖能力不受影响。结果初步表明藻酸钙与人骨髓基质干细胞体外生物相容性较好。      

Material properties and bone marrow stromal cells response to in situ crosslinkable RGD-functionlized lactide-co-glycolide scaffolds

In situ crosslinkable biomaterials with degradation profiles that can be tailored to a particular application are indispensable for treating irregularly shaped defects and for fabrication of shape-selective scaffolds. The objective of this work was to synthesize ultra low molecular weight functionalized PLA and PLGA macromers that can be grafted with bioactive peptides and crosslinked in situ to fabricate biodegradable functional scaffolds. In situ crosslinkable lactide-co-glycolide macromer (cMLGA; "c" for crosslinkable, "M" for macromer, and "LGA" for lactide-co-glycolide) was synthesized by anionic polymerization of lactide and glycolide monomers followed by condensation polymerization with fumaryl chloride. The cMLA (100% L-lactide) and cMLGA macromers formed porous crosslinked scaffolds with NVP as the crosslinker. The mass loss of the crosslinked cMLA and cMLGA was linear with incubation time in vitro (zero-order degradation) and the degradation rate depended on the ratio of lactide to glycolide. cMLGA scaffold with 1:1 lactide to glycolide ratio completely degraded after 4 weeks while the cMLA lost less than 40% of its initial mass after 35 weeks. When cMLA scaffold was functionalized with acrylated integrin-binding Ac-GRGD amino acid sequence, bone marrow stromal (BMS) cells attached and spread on the cMLA scaffold and exhibited focal-point cell adhesion. The mRNA expression levels of collagen-1alpha, osteonectin, and osteopontin for BMS cells seeded in the scaffolds with 1 and 5% Ac-GRGD were upregulated compared with those without Ac-GRGD. cMLGA is attractive as in situ crosslinkable macromer for fabrication of functional scaffolds with degradation characteristics that can be tailored to a particular application.     

Osteogenic differentiation and angiogenesis with cocultured adipose-derived stromal cells and bone marrow stromal cells

The purpose of this study was to determine the influence of cocultured adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation and angiogenesis of bone marrow stromal cells (BMSCs) as well as the underlying mechanism and the optimal ratio. Two in vitro coculture models, segregated cocultures using transwell and mixed cocultures, were employed to assess the indirect and direct effects of coculture respectively. Coculture was carried out for 14 days using 1 × 10⁵ BMSCs and ASCs of variable number. BMSCs, ASCs, or both were seeded in PLGA scaffold and implanted in the subcutaneous tissue of 25 nude mice for in vivo analysis of angiogenesis. To evaluate the orthotopic bone formation, critical size calvarial defects were created on 20 mice, and implanted with hydroxyapatite/β-tricalcium phosphate granules plus BMSCs, ASCs, or both. From both transwell and mixed coculture model, 1 × 10⁵ BMSCs cocultured with 0.5 × 10⁵ ASCs showed significantly greater osteogenic differentiation and mineralization than BMSCs alone. The mixed ASC/BMSC coculture at or above a ratio of 0.5/1 showed increased secretion of vascular endothelial growth factor (VEGF), and induced effective tube formation from human umbilical vein endothelial cells, which were comparable to ASCs. Cytokine profiling assay and gene expression study showed elevated levels of angiogenic factors VEGF and CXCL1, osteogenic factor Wnt5a as well as transforming growth factor (TGF)-βR1 and SMAD3 from BMSCs when cocultured with ASCs. After 5 weeks of implantation, polylactic-co-glycolic acid (PLGA)-ASCs-BMSCs had a number of vascular structures comparable to PLGA-ASCs and significantly greater than PLGA-BMSCs. Calvarial defects treated with ceramic/BMSCs/ASCs had greater area of repair and better reconstitution of osseous structure than the defects treated with ceramic/ASCs or ceramic/BMSCs after 10 weeks. In conclusion, ASCs added to BMSCs promoted osteogenesis and angiogenesis at the optimal ASC/BMSC ratio of 0.5/1.     

可注射性nHA/CS-BMSCs复合物修复兔股骨缺损的实验研究

目的评估可注射性纳米羟基磷灰石/壳聚糖材料(nHA/CS)复合骨髓基质干细胞(BMSCs)在促进兔股骨骨缺损修复中的作用。方法 18只成年新西兰大白兔双侧股骨外侧髁均建立骨缺损模型,造模后随机分为3组(1)实验组:18只兔右侧股骨缺损植入nHA/CS-BMSCs复合材料;(2)对照组:其中16只兔左侧股骨髁缺损植入单纯CS凝胶材料;(3)空白组:2只兔左侧骨缺损旷置,不植入任何材料。于第12周末取材,对缺损修复区组织行大体、影像学、形态计量学观察。实验组和对照组之间进行比较,观察该复合材料对骨缺损的修复效果。结果 18只兔均进入结果分析,X线侧位片及CT检查示实验组修复疗效优于对照组。对缺损修复区CT值测量分析显示实验组新骨形成多于对照组,差异有统计学意义(P0.05)。结论在促进骨缺损的修复中,nHA/CS-BMSCs复合材料疗效优于单纯CS材料的修复能力,具有确切的骨缺损修复能力,有良好的应用前景。     

Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.     

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.     

Smooth muscle-like tissues engineered with bone marrow stromal cells

Bone marrow-derived cells have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. Here we tested whether smooth muscle (SM)-like tissues can be created in vivo with bone marrow stromal cells (BMSCs). Cultured canine BMSCs, which expressed SM cell-specific markers including SM alpha-actin and SM myosin heavy chain, were seeded on three-dimensional, biodegradable polymer scaffolds and implanted into peritoneal cavity of athymic mice. The cell-scaffold constructs retrieved 4 weeks after implantation formed three-dimensional tissues. Immunohistochemical analyses showed that the tissue reconstructs expressed SM alpha-actin and SM myosin heavy chain. Masson's trichrome staining showed the presence of significant amounts of collagen in the tissue reconstructs. Cells labeled with a fluorescent tracer prior to implantation were still present in the tissue reconstructs 4 weeks after implantation. Non-seeded scaffolds (control groups) retrieved 4 weeks after implantation did not exhibit extensive tissue formation. This study demonstrates the potential of BMSCs as an alternative cell source for tissue engineering of SM.     

Growth and differentiation of bone marrow stromal cells on biodegradable polymer scaffolds: an in vitro study

A fundamental component of bone tissue engineering is an appropriate scaffold as a carrier for osteogenic cells. The aim of the study was to evaluate the response of human bone marrow stromal cells (BMSC) to scaffolds made of three biodegradable polymers: poly(L-lactide-co-ε-caprolactone) (poly(LLA-co-CL)), poly(L-lactide-co-1,5dioxepan-2-one) (poly(LLA-co-DXO)), and poly(L-lactide) (poly(LLA)). Cellular response was evaluated in terms of attachment, proliferation, and differentiation. SEM disclosed earlier cell attachment and better spreading on poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds than on poly(LLA) after 1 h. At 24 h and 14 days postseeding, BMSCs had spread well, forming multiple cellular layers on the scaffolds. Cell proliferation was higher on poly(LLA-co-CL) and on poly(LLA-co-DXO) than on poly(LLA) after 1 and 7 days. Cell growth cycles of BMSC were longer on the scaffolds than on coverslips. After 7 and 14 days cultivation on scaffolds, the expression of osteogenic markers such as ALP, Col I, OPN, and Runx2 were stimulated by BMSC, which indicating that poly(LLA-co-DXO), poly(LLA-co-CL), and poly(LLA) could support the osteogenic differentiation of BMSC in vitro. Poly(LLA-co-CL) and poly(LLA-co-DXO) promoted better attachment and growth of BMSC than poly(LLA). BMSC also retained their osteogenic differentiation potential, indicating biological activity of BMSC on the scaffolds. The promising results of this in vitro study indicate that these copolymers warrant further evaluation for potential application in bone tissue engineering.     

Centrifugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds

Bone marrow stromal cells (MSCs) are a promising cell source for a variety of tissue engineering applications, given their ready availability and ability to differentiate into multiple cell lineages. MSCs have been successfully used to create neotissue for cardiovascular, urological, and orthopedic reconstructive surgical procedures in preclinical studies. The ability to optimize seeding techniques of MSCs onto tissue engineering scaffolds and the ability to control neotissue formation in vitro will be important for the rational design of future tissue engineering applications using MSCs. In this study we investigated the effect of centrifugal force on seeding MSCs into a biodegradable polyester scaffold. MSCs were isolated and seeded onto porous scaffold sections composed of nonwoven polyglycolic acid mesh coated with poly(L-lactide-co-epsilon-caprolactone). Compared to standard static seeding techniques, centrifugal seeding increased the seeding efficiency by 38% (p < 0.007) and significantly improved cellular distribution throughout the scaffold. Overall, centrifugal seeding of MSCs enhances seeding efficiency and improves cellular penetration into scaffolds, making it a potentially useful technique for manipulating neotissue formation by MSCs for tissue engineering applications.