Atelocollagen Gel Research Articles

820 MICRO RNA29A RECRUITMENT AMELIORATES FIBROSIS INDUCED BY ANGIOTENSIN II VIA RAC1 MODULATION

Angiotensin II promotes renal fibrosis in chronic kidney disease via multiple intracellular pathways. How it regulates profibrotic genes is not fully understood. We hypothesized that miR29a, which is highly expressed in the kidney, may play a key role in angiotensin II-dependent signaling mediating fibrosis. Angiotensin II, oxidative stress, and transforming growth factor suppressed miR29a in distal tubule cells and changed the expression of profibrotic genes. Moreover, miR29a suppressed T-lymphoma invasion and metastasis inducing protein-1, a guanine nucleotide exchange factor, and inhibited the activity of the NADPH oxidase component, Rac1. This suggests a role for miR29a in oxidative stress. In angiotensin II-treated and salt-loaded uninephrectomized mice, miR29a expression was decreased and profibrotic genes such as matrix metalloproteinase 2 and collagens were enhanced. Induction of miR29a by atelocollagen gel attenuated kidney fibrosis in angiotensin II-treated uninephrectomized mice. Taken together, our data demonstrate that transforming growth factor β and oxidative stress, two distinct and well known downstream signaling cascades of Angiotensin II, share miR29a as a common molecule. miR29a may be a potent therapeutic target for renal fibrosis by decreasing the expression of profibrotic genes and reducing oxidative stress via suppressing T-cell lymphoma invasion and metastasis-inducing protein 1 and, in turn, Rac1 activity.

The therapeutic potential of ex vivo expanded CD133+ cells derived from human peripheral blood for peripheral nerve injuries

CD133(+) cells have the potential to enhance histological and functional recovery from peripheral nerve injury. However, the number of CD133(+) cells safely obtained from human peripheral blood is extremely limited. To address this issue, the authors expanded CD133(+) cells derived from human peripheral blood using the serum-free expansion culture method and transplanted these ex vivo expanded cells into a model of sciatic nerve defect in rats. The purpose of this study was to determine the potential of ex vivo expanded CD133(+) cells to induce or enhance the repair of injured peripheral nerves. Phosphate-buffered saline (PBS group [Group 1]), 10(5) fresh CD133(+) cells (fresh group [Group 2]), 10(5) ex vivo expanded CD133(+) cells (expansion group [Group 3]), or 10(4) fresh CD133(+) cells (low-dose group [Group 4]) embedded in atelocollagen gel were transplanted into a silicone tube that was then used to bridge a 15-mm defect in the sciatic nerve of athymic rats (10 animals per group). At 8 weeks postsurgery, histological and functional evaluations of the regenerated tissues were performed. After 1 week of expansion culture, the number of cells increased 9.6 ± 3.3-fold. Based on the fluorescence-activated cell sorting analysis, it was demonstrated that the initial freshly isolated CD133(+) cell population contained 93.22% ± 0.30% CD133(+) cells and further confirmed that the expanded cells had a purity of 59.02% ± 1.58% CD133(+) cells. However, the histologically and functionally regenerated nerves bridging the defects were recognized in all rats in Groups 2 and 3 and in 6 of 10 rats in Group 4. The nerves did not regenerate to bridge the defect in any of the rats in Group 1. The authors' results show that ex vivo expanded CD133(+) cells derived from human peripheral blood have a therapeutic potential similar to fresh CD133(+) cells for peripheral nerve injuries. The ex vivo procedure that can be used to expand CD133(+) cells without reducing their function represents a novel method for developing cell therapy for nerve defects in a clinical setting.

Evaluation of magnetic resonance imaging and clinical outcome after tissue-engineered cartilage implantation: prospective 6-year follow-up study

BackgroundAutologous chondrocyte implantation (ACI) is an important procedure when repairing cartilage defects of the knee. We previously reported several basic studies on tissue-engineered cartilage, and conducted a multicenter clinical study in 2009. In this clinical study, we evaluated the patients' clinical scores and MRI findings before and after tissue-engineered cartilage implantation, and compared the data obtained at 1year and approximately 6years post-implantation. MethodsFourteen patients who underwent implantation of tissue-engineered cartilage to repair cartilage defects of the knee were evaluated. Tissue-engineered cartilage was produced by culturing autologous chondrocytes three dimensionally in atelocollagen gel. The patients were evaluated clinically using the Lysholm score, and the original knee-function score at pre-implantation and at 1year and approximately 6years post-implantation. MRI scans were obtained at the same observation periods. A modified magnetic resonance observation of cartilage repair tissue (MOCART) system was used to quantify clinical efficacy based on the MRI findings. ResultsIn approximately 6years of follow-up, none of the 14 patients reported any subjective symptoms of concern. The mean Lysholm score and the original knee-function score (63.0 ± 10.1, 59.9 ± 5.7) significantly improved at 1year after implantation (86.4 ± 11.8, 94.1 ± 9.2), and were maintained until 6years after implantation (89.8 ± 6.2, 89.9 ± 11.2), although some patients showed deterioration of Lysholm and original knee scores between 1year post-implantation and the final follow-up. The mean MOCART score was 13.2 ± 12.0 pre-implantation, and 62.5 ± 24.7 at 1year and 70.7 ± 22.7 at approximately 6years post-implantation. The MOCART scores at 1year and 6years were significantly higher than the pre-implantation score, but there was no significant difference between the scores at 1 and 6years, indicating that the MRI results at 1year after implantation were maintained for the next 5years. ConclusionsThe clinical scores and MRI findings after implantation of tissue-engineered cartilage were improved at 1year after implantation and were maintained until 6years after implantation.

Potential of exogenous cartilage proteoglycan as a new material for cartilage regeneration

Although proteoglycan (PG) is one of the major components of cartilage matrices, its biological function is not fully elucidated. The objectives of this study were to investigate the proliferation and differentiation of chondrocytes embedded in atelocollagen gel with exogenous cartilage PG (PG-atelocollagen gel) in vitro, and also to evaluate the repair of cartilage defects by PG-atelocollagen gel in vivo. In the in vitro study, rabbit chondrocytes were cultured in the PG-atelocollagen gel. Cell proliferation and mRNA expression levels were measured, and gels were histologically evaluated. In the in vivo study, cultured PG-atelocollagen gel containing chondrocytes were transplanted into full-thickness articular cartilage defects in rabbit knees, and evaluated macroscopically and histologically. For the in vitro study, chondrocyte proliferation in 5.0 mg/ml PG-atelocollagen gel was enhanced, and the gene expression of Col2a1 and Aggrecan were decreased. In contrast, chondrocyte proliferation in 0.1 and 1.0 mg/ml PG-atelocollagen gel was not enhanced. The gene expression of Aggrecan in 0.1 and 1.0 mg/ml PG-atelocollagen gel was increased. For the in vivo study, the histological average total score of the 0.1 mg/ml PG-atelocollagen gel was significantly better than that of the group without PG. Although the appropriate concentration of PG has not been defined, this study suggests the efficacy of PG for cartilage repair.

Development of an injectable chitosan/marine collagen composite gel

A chitosan/marine-originated collagen composite has been developed. This composite gel was characterized and its biocompatibility, as well as an inflammatory reaction, was observed. The chitosan gel including N-3-carboxypropanoil-6-O-(carboxymethyl) chitosan of 3 mol%, 6-O-(carboxymethyl) chitosan of 62 mol% and 6-O-(carboxymethyl) chitin of 35 mol% was prepared and compounded with the salmon atelocollagen (SA) gel at different mixture ratios. The composite gels were injected subcutaneously in to the back of rats. The specimens were harvested for a histological survey as well as a tumor necrosis factor-alpha (TNF-α) assay by ELISA. The inflammatory cell infiltration and release of TNF-α were successively controlled low with the ratio of SA to chitosan at 10:90 or 20:80. The SA gel first, within 2 weeks, and then chitosan in the composite gel were slowly absorbed after implantation, followed by soft tissue formation. It is expected that this composite gel will be available as a carrier for tissue filler and drug delivery systems.

Efficacy of Bone Marrow Mononuclear Cells to Promote Bone Regeneration Compared With Isolated CD34+ Cells From the Same Volume of Aspirate

Autologous bone marrow mononuclear cell (BMMNC) transplantation is currently an emerging clinical treatment in the orthopedic as well as cardiovascular fields. It is believed that the therapeutic effect of the BMMNCs is due to neovascularization enhanced by the CD34(+) cells contained therein, which include endothelial progenitor cells. However, isolation of the CD34(+) cell fraction for clinical application has many disadvantages such as cost and invasiveness related to cell mobilization with cytokine. To investigate whether a purification step is in fact necessary for bone regeneration, we separated BMMNCs, CD34(+), and CD34(-) cells from the same initial volume of rabbit bone marrow aspirates. We then transplanted them back into a femoral bone defect of the same rabbit together with atelocollagen gel and basic fibroblast growth factor (bFGF) and evaluated neovascularization and bone regeneration up to 8 weeks after transplantation. The greatest potential for neovascularization and bone regeneration medicated by cells from the same volume of bone marrow aspirate was found in the BMMNC group. Although purified CD34(+) cells might be an ideal cell source, BMMNCs could be a practical and feasible cell source for bone regeneration in present clinical settings with limited cost, availability of materials, and technical issues for transplantation.

The effect of microRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel

The objective of this study was to investigate the effect of microRNA-21 (miR-21) on the proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Articular cartilage was harvested aseptically from the knee and hip joints of rats. In the experimental group, double-stranded miR-21 was transfected into the chondrocytes, and in the control group, scrambled siRNA was used. After that, chondrocytes were cultured in atelocollagen gel for 3 weeks. At 1, 2, and 3 weeks after transfection, the cell numbers were counted, and the expression levels of Col2a1 and aggrecan were measured by real-time PCR. Histological analysis by toluidine blue staining was performed at 3 weeks. The cell number in the experimental group rapidly increased compared to the control group. The expression levels of Col2a1 and aggrecan in the experimental group were higher than in the control. Histological analysis revealed that many more cells with a metachromatic stain were present in the experimental group than in the control. This study demonstrated that miR-21 promotes high proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel.

Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan

BackgroundNew tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees. MethodsAt six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months. ResultsThe Lysholm score significantly increased from 60.0 ± 13.7 points to 89.8 ± 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases. ConclusionsWe concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

Effect of Monosaccharides Composing Glycosaminoglycans on Type 2 Collagen Accumulation in a Three-Dimensional Culture of Chondrocytes

The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes.

The application of atelocollagen gel in combination with porous scaffolds for cartilage tissue engineering and its suitable conditions

For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of cartilage matrices. The CS was soft with a Young's modulus of less than 1 MPa, whereas the porous PLLA was very rigid with a Young's modulus of 10 MPa. Although the constructs with the CS shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes, the cartilage regeneration was improved in inverse proportion to the passage numbers among passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.

Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult to achieve full functional recovery in humans following extended peripheral nerve lesions. In this study, the authors examined the use of blood-derived CD133(+) cells in promoting the repair of peripheral nerve defects. The authors transplanted phosphate-buffered saline (control), mononuclear cells, or CD133(+) cells embedded in atelocollagen gel into a silicone tube that was used to bridge a 15-mm defect in the sciatic nerve of athymic rats (12 animals in each group). At 8 weeks postsurgery, molecular, histological, and functional evaluations were performed in regenerated tissues. The authors found that sciatic nerves in which a defect had been made were structurally and functionally regenerated within 8 weeks after CD133(+) cell transplantation. From macroscopic evaluation, massive nervelike tissues were confirmed only in rats with CD133(+) cell transplantation compared with the other groups. Morphological regeneration in the samples after CD133(+) cell transplantation, as assessed using toluidine blue staining, was enhanced significantly in terms of the number of myelinated fibers, axon diameter, myelin thickness, and percentage of neural tissue. Compound muscle action potentials were observed only in CD133(+) cell-treated rats. Furthermore, it was demonstrated that the transplanted CD133(+) cells differentiated into Schwann cells by 8 weeks after transplantation. The results show that CD133(+) cells have potential for enhancement of histological and functional recovery from peripheral nerve injury. This attractive cell source could be purified easily from peripheral blood and could be a feasible autologous candidate for peripheral nerve injuries in the clinical setting.

Chondrogenic differentiation of human umbilical cord blood-derived multilineage progenitor cells in atelocollagen

For successful stem cell-based therapy, not only are alternative good cell sources needed but appropriate scaffolds are key factors. The main purpose of this study was to evaluate the multipotentiality of multilineage progenitor cells (MLPC) and assess the three-dimensional cultivation and chondrogenic differentiation of MLPC in atelocollagen gel for application of tissue-engineered cartilage constructs. MLPC, human umbilical cord blood-derived clonal cell lines, from BioE Inc. were used. Immunophenotypes of MLPC were characterized using a fluorescence-activated cell sorter (FACS). In vitro differentiation potentials into osteogenic, adipogenic and chondrogenic lineages were examined. Differentiated cells were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR), histologic and immunofluorescence staining. RESULTS Clonogenic MLPC maintained immunophenotypes with specific surface markers of mesenchymal stromal cells (MSC). The osteogenic and adipogenic potentials of MLPC were demonstrated by quantitative real-time PCR, alkaline phosphates activity and Oil Red O staining. Furthermore, MLPC were successfully differentiated into chondrocytes in atelocollagen gel, which was confirmed by RT-PCR and immunofluorescence staining for type II collagen protein. Whenever MSC are considered for the treatment of cartilage defects, a variety of scaffolds have been utilized as successful carriers for cell delivery. Our results suggest that MLPC can serve as an alternative source for stem cell-based therapy and transplantation. The chondrogenic potential of MLPC in atelocollagen could be suitable for cartilage tissue engineering.

Transplantation of Tissue‐Engineered Osteochondral Plug Using Cultured Chondrocytes and Interconnected Porous Calcium Hydroxyapatite Ceramic Cylindrical Plugs to Treat Osteochondral Defects in a Rabbit Model

The purpose of this study was to evaluate the macroscopic and histological results of transplanting a tissue-engineered composite plug made of tissue-engineered cartilage and interconnected porous calcium hydroxyapatite ceramics (IP-CHA) with a very high porosity of 94.9% to treat osteochondral defects. Twelve 12-week-old male Japanese white rabbits were used. Fresh articular cartilage slices were taken, and isolated chondrocytes (2 x 10(6) cells) were embedded in atelocollagen gel. They were seeded on the top of IP-CHA plugs and cultured for 2 weeks. These tissue-engineered composite plugs were transplanted into the osteochondral defects in the patellar grooves (the experimental group). In the control group, the defects were treated with composite plugs without chondroytes. Twelve weeks after transplantation in the experimental group, the defects were repaired with cartilage-like tissue with good subchondral bone formation histologically. Histological scores in the experimental group were significantly better than those in the control group. This study clearly showed the defects that had been treated with tissue-engineered composite plugs.

Optimal Combination of Soluble Factors for Tissue Engineering of Permanent Cartilage from Cultured Human Chondrocytes

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2(12) = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.

Effects of Atelocollagen Gel Containing Bone Marrow-Derived Stromal Cells on Repair of Osteochondral Defect in a Dog

To clarify the contribution of autologous transplantation of mesenchymal stromal cells (MSCs), an atelocollagen gel containing or not containing fluorescently-labeled canine MSCs was transplanted into an osteochondral defect which did not repair spontaneously and the histological repair of the defect was compared. Although an early repair of the cartilage was not observed in either defect, the reproduction of subchondral bone was remarkable in the MSCs-implanted defect. Moreover, in 2 weeks after operation, the implanted MSCs were located in the deeper regions of the defect, suggesting the differentiation of osteoblasts. There was a possibility that the movement of the implanted MSCs was due to an increase in intra-articular pressure from postoperative inflammation.

Hypoxia promotes chondrocytic proliferation and cartilage matrix synthesis: transplantation of autologus chondrocytes embedded in an atelocollagen gel

Hypoxia promotes chondrocytic proliferation and cartilage matrix synthesis: transplantation of autologus chondrocytes embedded in an atelocollagen gel

Mesenchymal progenitor cells derived from chorionic villi of human placenta for cartilage tissue engineering

Human mesenchymal stem cells are currently being studied extensively because of their capability for self-renewal and differentiation to various connective tissues, which makes them attractive as cell sources for regenerative medicine. Herein we report the isolation of human placenta-derived mesenchymal cells (hPDMCs) that have the potential to differentiate into various lineages to explore the possibility of using these cells for regeneration of cartilage. We first evaluated the chondrogenesis of hPDMCs in vitro and then embedded the hPDMCs into an atelocollagen gel to make a cartilage-like tissue with chondrogenic induction media. For in vivo assay, preinduced hPDMCs embedded in collagen sponges were subcutaneously implanted into nude mice and also into nude rats with osteochondral defect. The results of these in vivo and in vitro studies suggested that hPDMCs can be one of the possible allogeneic cell sources for tissue engineering of cartilage.

Repair of Osteochondral Defect With Tissue-Engineered Chondral Plug in a Rabbit Model

The purpose of this study was to evaluate the macroscopic and histologic results of transplanting a tissue-engineered chondral plug made of atelocollagen sponge and PLLA mesh to treat osteochondral defects. Controlled experimental study. Twelve-week-old male Japanese white rabbits were used. Fresh articular cartilage slices were taken from the humeral head, and isolated chondrocytes were embedded in atelocollagen gel which does not have antigenic portions of collagen (2.0 x 10(6) cells/mL). They were seeded on the top of the atelocollagen sponge/PLLA mesh composite and cultured for 2 weeks. The culture medium was changed every 3 days and L-ascorbic acid (50 microg/mL) was added every 2 days. Culturing the composites for 2 weeks produced tissue-engineered chondral plugs. These tissue-engineered chondral plugs (4-mm diameter, 4-mm thick) were transplanted into the osteochondral defects (4 mm diameter, 4 mm deep) in the patellar grooves of the same rabbits from which the chondrocytes had been harvested (the experimental group). In the control group, the defects were treated with the plugs without chondrocytes. The rabbits were killed 4 and 12 weeks after transplantation. The repaired tissues were evaluated macroscopically and histologically, and analyzed immunohistochemically for expression of type II collagen. Four weeks after transplantation in the experimental group, the defects were partially repaired with cartilage-like tissue with good subchondral bone formation. Twelve weeks after transplantation, the defects were repaired with hyaline cartilage-like tissue densely stained by Safranin O. Well-organized subchondral bone formation was also observed. In the control group, the defects were covered with only soft fibrous tissue at 4 and 12 weeks macroscopically. Immunohistochemically, type II collagen was detected in about 90% of the repaired area. Histologic scores in the experimental group were significantly higher than those in the control group at both 4 and 12 weeks after transplantation. This study shows that the defects treated with tissue engineered chondral plug developed type II collagen in about 90% of the repaired area. The transplantation of a tissue-engineered chondral plug will be one option for treating osteochondral defects. The next step in testing our hypothesis is to evaluate the repaired tissue biomechanically and biochemically over a longer period of time.

Process design of chondrocyte cultures with monolayer growth for cell expansion and subsequent three-dimensional growth for production of cultured cartilage

The subculture of rabbit chondrocytes with serial passaging was carried out for cell expansion on a collagen-coated surface, and the morphological transition of round-shaped cells to spindle-shaped ones was examined. The observation of cytoskeletal formation by staining F-actin and vinculin supported the view that the round-shaped cells were in the process of differentiation with immature stress fibers relating to less cellular polarity. The cellular morphology was estimated in terms of the distribution of roundness, R(C), during the subculturing on the collagen substrate. The frequency of the number of round-shaped cells, which was defined as the ratio of the number of cells with R(C) >0.9 against the total cell number, was correlated in a logarithmic formula with the number of population doublings during the subcultures. Kinetic models were adopted for the process design of the combined culture of chondrocytes with monolayer growth on the collagen substrate and subsequent three-dimensional growth in Atelocollagen gel, employing the boundary conditions based on the population balance between differentiated and dedifferentiated cells. The combined culture was performed successfully according to the process design scheduled as monolayer growth for 240 h and three-dimensional growth for 264 h, the number of seed cells being 68% of that in the conventional culture for 504 h where monolayer growth for cell expansion was not included.

Neovascularization and bone regeneration by implantation of autologous bone marrow mononuclear cells

We examined whether transplantation of autologous bone marrow mononuclear cells (BM-MNCs) can augment neovascularization and bone regeneration of bone marrow in femoral bone defects of rabbits. Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. To evaluate the in vivo effect of implanted BM-MNCs, we created bone defects in the rabbit medial femoral condyle, and implanted into them 5×10 6 fluorescent-labeled autologous BM-MNCs together with gelatin microspheres containing 10 μg bFGF on an atelocollagen gel scaffold. The four experimental groups, which were Atelocollagen gel (Col), Col+5×10 6 BM-MNCs, Col+10 μg bFGF, and Col+5×10 6 BM-MNCs+10 μg bFGF, were implanted into the sites of the prepared defects using Atelocollagen gel as a scaffold. The autologous BM-MNCs expressed CD31, an endothelial lineage cell marker, and induced efficient neovascularization at the implanted site 2 weeks after implantation. Capillary density in Col+BM-MNCs+bFGF was significantly large compared with other groups. This combination also enhanced regeneration of the bone defect after 8 weeks to a significantly greater extent than either BM-MNCs or bFGF on their own. In summary, these findings demonstrate that a combination of BM-MNCs and bFGF gelatin hydrogel enhance the neovascularization and the osteoinductive ability, resulting in bone regeneration.