Bone Marrow Stromal Cell Sheets Research Articles

Scaffold-Free tissue engineering with aligned bone marrow stromal cell sheets to recapitulate the microstructural and biochemical composition of annulus fibrosus

Current tissue engineering strategies through scaffold-based approaches fail to recapitulate the complex three-dimensional microarchitecture and biochemical composition of the native Annulus Fibrosus tissue. Considering limited access to healthy annulus fibrosus cells from patients, this study explored the potential of bone marrow stromal cells (BMSC) to fabricate a scaffold-free multilamellar annulus fibrosus-like tissue by integrating micropatterning technologies into multi-layered BMSC engineering. BMSC sheet with cells and collagen fibres aligned at ~30° with respect to their longitudinal dimension were developed on a microgroove-patterned PDMS substrate. Two sheets were then stacked together in alternating directions to form an angle-ply bilayer tissue, which was rolled up, sliced to form a multi-lamellar angle-ply tissue and cultured in a customized medium. The development of the annulus fibrosus-like tissue was further characterized by histological, gene expression and microscopic and mechanical analysis. We demonstrated that the engineered annulus fibrosus-like tissue with aligned BMSC sheet showed parallel collagen fibrils, biochemical composition and microstructures that resemble the native disk. Furthermore, aligned cell sheet showed enhanced expression of annulus fibrosus associated extracellular matrix markers and higher mechanical strength than that of the non-aligned cell sheet. The present study provides a new strategy in annulus fibrosus tissue engineering methodology to develop a scaffold-free annulus fibrosus-like tissue that resembles the microarchitecture and biochemical attributes of a native tissue. This can potentially lead to a promising avenue for advancing BMSC-mediated annulus fibrosus regeneration towards future clinical applications.

Implantation of Bone Marrow Stromal Cell Sheets Derived from Old Donors Supports Bone Tissue Formation.

The purpose of this study was to evaluate the osteogenesis ability of osteogenic matrix cell sheets (OMCS) derived from old donor cells. Bone marrow stromal cells (BMSC) were obtained from young (7-week-old) and old (1-year-old) Fischer344 rats donors and cultured with modified Eagle's medium (MEM group) alone or containing dexamethasone (Dex; 10nM) and ascorbic acid phosphate (AscP; 0.28mM) (Dex/AscP group). We prepared four in vitro experimental groups: (1) young MEM, (2) young Dex/AscP, (3) old MEM and (4) old Dex/AscP. Cell proliferation and osteogenic marker mRNA expression levels were evaluated in vitro. To assess bone formation in vivo, the cells of each group were combined with beta tricalcium phosphate (TCP) disks followed by implantation in recipient rats. The in vitro study showed significant differences in the mRNA expression of osteocalcin, ALP, and BMP2 between MEM and Dex/AscP groups. Bone formation following implantation was observed upon histological analyses of all groups. TCP combined with OMCS (OMCS/TCP group) resulted in enhanced bone formation compared to that following combination with BMSC (BMSC/TCP). The osteocalcin content of the OMCS/TCP group 4weeks after implantation was significantly higher than that in the BMSC/TCP construct for both young and old donors. The present study clearly indicated that OMCS could be generated from BMSCs of old as well as young donors using a mechanical retrieval method. Thus, through its usage of OMCS, this method may represent a potentially effective therapeutic option for cell-based therapy in elderly patients.

Bone marrow stromal cell sheets may promote axonal regeneration and functional recovery with suppression of glial scar formation after spinal cord transection injury in rats.

OBJECTIVE Transplantation of bone marrow stromal cells (BMSCs) is a theoretical potential as a therapeutic strategy in the treatment of spinal cord injury (SCI). Although a scaffold is sometimes used for retaining transplanted cells in damaged tissue, it is also known to induce redundant immunoreactions during the degradation processes. In this study, the authors prepared cell sheets made of BMSCs, which are transplantable without a scaffold, and investigated their effects on axonal regeneration, glial scar formation, and functional recovery in a completely transected SCI model in rats. METHODS BMSC sheets were prepared from the bone marrow of female Fischer 344 rats using ascorbic acid and were cryopreserved until the day of transplantation. A gelatin sponge (GS), as a control, or BMSC sheet was transplanted into a 2-mm-sized defect of the spinal cord at the T-8 level. Axonal regeneration and glial scar formation were assessed 2 and 8 weeks after transplantation by immunohistochemical analyses using anti-Tuj1 and glial fibrillary acidic protein (GFAP) antibodies, respectively. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan scale. RESULTS The BMSC sheets promoted axonal regeneration at 2 weeks after transplantation, but there was no significant difference in the number of Tuj1-positive axons between the sheet- and GS-transplanted groups. At 8 weeks after transplantation, Tuj1-positive axons elongated across the sheet, and their numbers were significantly greater in the sheet group than in the GS group. The areas of GFAP-positive glial scars in the sheet group were significantly reduced compared with those of the GS group at both time points. Finally, hindlimb locomotor function was ameliorated in the sheet group at 4 and 8 weeks after transplantation. CONCLUSIONS The results of the present study indicate that an ascorbic acid-induced BMSC sheet is effective in the treatment of SCI and enables autologous transplantation without requiring a scaffold.

Effectiveness of Bone Marrow Stromal Cell Sheets in Maintaining Random-Pattern Skin Flaps in an Experimental Animal Model

Bone marrow stromal cells can be applied therapeutically to enhance angiogenesis; however, the use of bone marrow stromal cell suspensions reduces efficiency because of low-level attachment. The authors hypothesized that bone marrow stromal cell sheets would facilitate cell fixation, thus enhancing angiogenesis. The authors investigated flap survival area and enhancement of angiogenic factors in a rat random-pattern skin flap model after application of bone marrow stromal cell sheets. Bone marrow stromal cell sheets (prepared from 7-week-old rat femurs) were cultured under four different hypoxic conditions. Sheets with the highest angiogenic potential, determined by an in vitro pilot study, were injected into subcutaneous layers of the rat dorsum (bone marrow stromal cell sheet group). A control group (phosphate-buffered saline only) was included. On day 2 after injection, caudally based random-pattern skin flaps (12 × 3 cm) were elevated. On day 7 after elevation, surviving skin flap areas were measured. Skin samples were harvested from each flap and gene expression levels of vascular endothelial growth factor and basic fibroblast growth factor were measured by quantitative real-time polymerase chain reaction. Skin flap survival area (71.6 ± 2.3 percent versus 51.5 ± 3.3 percent) and levels of vascular endothelial growth factor and basic fibroblast growth factor were significantly higher in the bone marrow stromal cell sheet group than in the control group (p < 0.05). Implantation of bone marrow stromal cell sheets increased the survival area of random-pattern skin flaps. Expression of angiogenic factors may have contributed to the increased flap survival.

Delayed minimally invasive injection of allogenic bone marrow stromal cell sheets regenerates large bone defects in an ovine preclinical animal model.

Cell-based tissue engineering approaches are promising strategies in the field of regenerative medicine. However, the mode of cell delivery is still a concern and needs to be significantly improved. Scaffolds and/or matrices loaded with cells are often transplanted into a bone defect immediately after the defect has been created. At this point, the nutrient and oxygen supply is low and the inflammatory cascade is incited, thus creating a highly unfavorable microenvironment for transplanted cells to survive and participate in the regeneration process. We therefore developed a unique treatment concept using the delayed injection of allogenic bone marrow stromal cell (BMSC) sheets to regenerate a critical-sized tibial defect in sheep to study the effect of the cells' regeneration potential when introduced at a postinflammatory stage. Minimally invasive percutaneous injection of allogenic BMSCs into biodegradable composite scaffolds 4 weeks after the defect surgery led to significantly improved bone regeneration compared with preseeded scaffold/cell constructs and scaffold-only groups. Biomechanical testing and microcomputed tomography showed comparable results to the clinical reference standard (i.e., an autologous bone graft). To our knowledge, we are the first to show in a validated preclinical large animal model that delayed allogenic cell transplantation can provide applicable clinical treatment alternatives for challenging bone defects in the future.

Application of cell sheet technology to bone marrow stromal cell transplantation for rat brain infarct.

Bone marrow stromal cells (BMSC) transplantation enhances functional recovery after cerebral infarct, but the optimal delivery route is undetermined. This study was aimed to assess whether a novel cell-sheet technology non-invasively serves therapeutic benefits to ischemic stroke. First, the monolayered cell sheet was engineered by culturing rat BMSCs on a temperature-responsive dish. The cell sheet was analysed histologically and then transplanted onto the ipsilateral neocortex of rats subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their behaviours and histology were compared with those in the animals treated with direct injection of BMSCs or vehicle over 4 weeks post-transplantation. The cell sheet was 27.9 ± 8.0 μm thick and was composed of 9.8 ± 2.4 × 105 cells. Cell sheet transplantation significantly improved motor function when compared with the vehicle-injected animals. Histological analysis revealed that the BMSCs were densely distributed to the neocortex adjacent to the cerebral infarct and expressed neuronal phenotype in the cell sheet-transplanted animals. These findings were almost equal to those for the animals treated with direct BMSC injection. The attachment of the BMSC sheet to the brain surface did not induce reactive astrocytes in the adjacent neocortex, although direct injection of BMSCs profoundly induced reactive astrocytes around the injection site. These findings suggest that the BMSCs in cell sheets preserve their biological capacity of migration and neural differentiation. Cell-sheet technology may enhance functional recovery after ischaemic stroke, using a less invasive method. Copyright © 2014 John Wiley & Sons, Ltd.

Regeneration of Periosteum by Human Bone Marrow Stromal Cell Sheets

The presence of a functional periosteum accelerates healing in bone defects by providing a source of progenitor cells that aid in repair. We hypothesized that bone marrow stromal cell (BMSC) sheets could be used to engineer functional periosteal tissues. BMSCs were cultured to hyperconfluence and produced sufficient extracellular matrix to form robust tissue sheets. The sheets were wrapped around calcium phosphate pellets and implanted subcutaneously in mice for 8 weeks. Histologic comparisons were made between calcium phosphate samples with and without BMSC sheet wraps. Bone and periosteum formation were analyzed through tissue morphology and tissue-specific protein expression. Calcium phosphate pellets wrapped in BMSC sheets regenerated a bone-like tissue, but pellets lacking the cell sheet wrap did not. The bone-like tissue seen on the calcium phosphate scaffolds wrapped with the BMSC sheets was enclosed within a periosteum-like tissue characterized morphologically and through expression of periostin. These data indicate that cell sheet technology has potential for regenerating a functional periosteum-like tissue that could aid in future orthopedic therapy.

Periodontal tissue in a bio-implant by periodontal ligament cells sheet and bone marrow stromal cells sheet

To fabricate the bio-implant supported by regenerated periodontal tissue utilizing periodontal ligament cells (PDLC)-bone marrow stromal cells (BMSC) sheet and natural root. Premolars of 2 beagle dogs were extracted to prepare the implanted area. Autologous tooth roots were carved into cylinders. PDLC and BMSC separated from beagle dogs were cultured into cell sheets in medium. Tooth roots were wrapped by one type of cell sheets or both to fabricate bio-implant and divided into four groups, tooth roots were wrapped by PDLC sheets and BMSC sheets successively (2 samples each dog), tooth roots were wrapped by PDLC sheets alone (2 samples each dog), tooth roots were wrapped by BMSC sheet alone (2 samples each dog), tooth roots without cell sheet (1 sample each dog). The implants were implanted into the mandibles. The mandibles were dissected 12 weeks later, sliced and stained by HE and Masson dyes for histological examination. In the PDLC cell sheet/root implants, histological examination revealed that new periodontal-like tissue including cementum, periodontium and alveolar bone was regenerated.In the BMSC implants, tooth ankylosis was observed. PDLC sheet and natural root can be used to fabricate bio-implant. PDLC sheet could promote periodontal regeneration.

A Preliminary Study on the Application of Bone Marrow Stromal Cell Sheet on the Formation of Functional Tissue-Engineered Bone in Dogs

The aim of this study was to construct functional tissue-engineered bone in dogs using cell sheet engineering, a new technique to gain and transfer seed cells. Demineralized bone matrixes, prepared from homologous bone, were coated with recombinant human bone morphogenetic protein-2. Bone marrow stromal cells (BMSCs) were isolated and subcultured. Osteogenic-induced BMSCs were incubated in a temperature-responsive culture dish to form the BMSC sheet. The complex of demineralized bone matrix, recombinant human bone morphogenetic protein-2, and BMSCs wrapped with BMSC sheets was implanted around the blood vessels of the latissimus dorsi muscle in the experimental side, and the same complex without BMSC sheets was implanted around the blood vessels of the latissimus dorsi muscle on the other side as a control. At 4, 8, 12, and 16 weeks after implantation, the implants were removed for radiographic evaluation, descriptive histologic observation, and histologic quantitative analysis. Radiographic analysis showed that the optical density of the tissue-engineered bone on the 2 sides increased with time. However, the optical density of the experimental side was significantly greater than that of the control side at the same points. Sixteen weeks after implantation, mature lamellar bone was formed in the experimental side, with red bone marrow in the bone marrow cavity. In contrast, the control side exhibited significantly less lamellar bone. Histologic quantitative analysis showed that the experimental side exhibited significantly more bone per area compared with the control side. BMSC sheet engineering may be useful to construct functional tissue-engineered bone.

Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis: A rabbit model

Effective methods to shorten the treatment period of distraction osteogenesis (DO) are needed. To investigate whether injections of osteogenic bone marrow stromal cell (BMSC) sheet fragments could be used to facilitate new bone formation during DO, 30 rabbits underwent bilateral mandibular osteotomy and their mandibles were lengthened at a rate of 0.75 mm/12 h for 6 days after a 5-day latency period. There were three treatment groups (n = 10 for each group): Serum-free medium, dissociated BMSCs, and BMSC sheet fragments. A local injection was conducted with a needle directly into the distracted areas immediately after distraction. Rabbits were sacrificed for examination at 3 and 6 weeks after injection. Gross examination, radiographic evaluation, and micro-CT scanning indicated a significant increase in bony union in the BMSC sheet fragment group, compared with the medium group and the dissociated cell group. The histomorphometric analysis showed more intensive bone formation in the sheet fragment group than the other two groups at each time point. Additionally, the peak load was significantly higher in the fragment group than those in the others. The results show that injection of BMSC sheet fragments promotes bone formation in DO and indicate a promising approach to shorten the treatment period of osteodistraction.

Abstract 117: Therapeutic Impact of Direct Cell Sheet Transplantation Derived from Bone Marrow Stromal Cells for Cerebral Infarct in Rats

Background and Purpose - Less-invasive and efficient donor cell delivery is quite important for the clinical application of cell transplantation therapy. Recently, cell sheet technology has gained a great interest as a novel tissue engineering technology, but there are no studies that evaluate the validity of the bone marrow stromal cell (BMSC) sheets as the donor for cerebral infarct. The aim of this study is to assess whether the BMSC sheet can be an alternative transplantation technique to promote functional recovery after cerebral infarct. Methods - The BMSC were harvested from green fluorescence protein (GFP)-transgenic rat and were cultured on a temperature-responsive culture dishes. The mono-layered BMSC sheet was detached by changing the temperature of culture dishes, and was histologically analyzed. Next, the SD rats were subjected to permanent middle cerebral artery occlusion and were divided into 3 experimental groups. Thus, the vehicle or BMSC suspension was stereotactically transplanted into the ipsilateral striatum, or the BMSC sheet was directly transplanted onto the ipsilateral intact neocortex at 7 days after the insult (n=9, 9, 7, respectively). Motor function was assessed for 28 days after transplantation. Finally, histological analysis was performed. Results - BMSC sheet was composed of 9.8 ± 2.4 x10 5 cells (n=10). Stereotactic injection of BMSC significantly enhanced the recovery of motor function after cerebral infarct. Likewise, non-invasive cell sheet transplantation also significantly promoted functional recovery. The therapeutic effect was comparable to stereotactic cell transplantation. Fluorescence immunohistochemistry revealed that the GFP-positive cells were densely distributed in the dorsal neocortex adjacent to cerebral infarct in both groups. Double fluorescence immunohistochemistry demonstrated that the majority of engrafted GFP-positive cells were positive for NeuN and morphologically simulated the neurons in both groups. The density of reactive astrocytes in the ipsilateral striatum was less pronounced in cell sheet transplantation group than in stereotactic transplantation group. Conclusion - These findings strongly suggest that cell sheet technology would make it possible to non-invasively deliver the BMSC into the infarct brain and to yield significant therapeutic effects, when compared with stereotactic injection of cell suspension. The technology may be a clinically valuable and less-invasive construct for non-invasive and efficient cell delivery to regenerate the infarct brain.

Enhancing bone formation by transplantation of a scaffold‐free tissue‐engineered periosteum in a rabbit model

The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP). We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous β-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model. The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site. The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings.

Autogenous Bone Marrow Stromal Cell Sheets-Loaded mPCL/TCP Scaffolds Induced Osteogenesis in a Porcine Model of Spinal Interbody Fusion

This study was designed to investigate whether a tissue-engineered construct composed of autogenous cell sheets and a polycaprolactone-based bioresorbable scaffold would enhance bone regeneration and spinal interbody fusion in a large animal model. Porcine-derived autogenous bone marrow stromal cells (BMSCs) cultured into multilayered cell sheets were induced into osteogenic differentiation with dexamethasone, l-ascorbic acid, and β-glycerol phosphate. These cell sheets were assembled with bioresorbable scaffolds made from medical-grade poly(epsilon-caprolactone) incorporating 20% β-tricalcium phosphate (mPCL/TCP) as tissue-engineered BMSC constructs. L2/3, L4/5 discectomies and decortication of the vertebral end plates were performed on 16 SPF Yorkshire pigs through an anterolateral approach. The tissue-engineered BMSC constructs were transplanted into the prepared intervertebral disc spaces of half of the pigs (n = 8), whereas cell-free mPCL/TCP served as controls in the remaining pigs. New bone formation and spinal fusion were evaluated at 3 and 6 months using microcomputed tomography, histology, fluorochrome bone labeling, and biomechanical testing. New bone formation was evident as early as 3 months in the BMSC group. At 6 months, bony fusion was observed in >60% (5/8) of segments in the BMSC group. None of the control animals with cell-free scaffold showed fusion at both time points. Biomechanical evaluation further revealed a significantly increased segmental stability in the BMSC group compared with the cell-free group at 6 months postimplantation (p < 0.01). These findings suggest that mPCL/TCP scaffolds loaded with in vitro differentiated autogenous BMSC sheets could induce bone formation and interbody fusion. This in turn resulted in enhanced segmental stability of the lumbar spine.

Ectopic Bone Induction by BMP-loaded Collagen Scaffold and Bone Marrow Stromal Cell Sheet

19 INTRODUCTION Bone inductive therapies have been developed using tissue engineering with three essential factors, i.e., osteogenic cells, osteoinductive growth factor and a scaffold for bone-forming cells. Bone marrow stromal cells (BMSCs) include multipotent mesenchymal stem cells. BMSCs could differentiate into several cell-types including osteoblasts , and implantation of BMSCs with various biological scaffolds enables to induce a new bone in vivo. Grafting with calcium alginate to alveolar bone defects in dogs accelerated bone regeneration. Bruder et al. reported that the combined implantation with hydroxyapatite/β-tricalcium phosphate ceramics and bone marrow-derived mesenchymal stem cells facilitated bone formation: They insisted that a ceramic scaffold carrying cell source could be primarily biocompatible and be able to provide mechanical stiffness compared to the ceramics scaffold per se. A biological scaffold may play an Ectopic Bone Induction by BMP-loaded Collagen Scaffold and Bone Marrow Stromal Cell Sheet

Constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells sheet and PLGA internal support: experimental study in bioreactor

To explore the feasibility of constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells (BMSCs) sheet and PLGA internal support. Rabbit BMSCs were expanded and induced by transforming growth factor-1 to improve chondrocyte phenotype of BMSCs. BMSCs sheets were obtained by continuous culture and wrapped the PGLA scaffold in the shape of cylinder. The constructs were incubated in spinner flask for 8 weeks and cartilage formation was investigated by gross inspection, histology, glycosaminoglycan and mechanical strength content. After in vitro culture, cartilage like tissue in cylindrical shape had been regenerated successfully. Stiff, shiny, pearly opalescence tissues were observed. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix, cells stationed in the lacunae could be noticed clearly. Safranin-O staining on the sections showed homogenous and positive red staining, which demonstrated that the engineered tissue was rich in proteoglycans. Based on the cell sheet and internal support strategy, trachea-like cartilage in cylindrical shape could be successfully fabricated which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea-cartilage loss encountered in clinical practice.

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5×5×4 mm 3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10×10×4 mm 3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

Assembly of bone marrow stromal cell sheets with knitted poly (L‐lactide) scaffold for engineering ligament analogs

The current cell seeding technique has several disadvantages, such as low efficiency of cell attachment to scaffolds and the limited strength of cell-gel composite adhesion to scaffold. These problems warrant further study to improve the assembly of cell to scaffold. Therefore this study aims to fabricate a bone marrow stromal cells (bMSCs) sheet and assemble it on a knitted poly (L-lactide) (PLLA) scaffold for engineering ligament analogs. bMSCs were cultured to form a cell sheet in the presence of ascorbic acid. Once a sheet of bMSCs was obtained, it was assembled onto the knitted scaffold by a wrapping technique. Then the assembled structure was held in place in a spinner flask for 4 weeks. The macromorphology, histology, and biomechanics of the grafts were evaluated. The composite of cell sheet/PLLA scaffold constructs had transformed into tissuelike ligament analogs. Immunohistochemical analysis showed that the components of the analogs were similar to that of ligament tissues, consisting primarily of collagen type I and small amount of collagen type III and tenascin. The failure force of the cell/scaffold assembly under tension (46.68+/-2.29 N) was higher than that of the scaffold group (43.58+/-2.41 N; p<0.05), but tensile stiffness of the cell/scaffold group (20.6+/-1.417 N/mm) was significantly lower than that of the scaffold group (27.6+/-1.449 N/mm; p<0.05). These data showed that the incorporation of bMSCs sheet onto the PLLA scaffold could make the analog stronger and more stretchable. Therefore the approach of assembling bMSCs sheet onto knitted PLLA scaffold is promising for producing tissuelike and functional ligament analogs under dynamic fluid situation for the purpose of anterior cruciate ligament (ACL) reconstruction.