【Abstract】 Objective To review the recent progress of BMSCs acting as seeding cell for tissue engineeredcartilage. Methods The recent ten years l iterature about BMSCs acting as seeding cell for tissue engineered cartilage was extensively reviewed. Results Scaffold provided an optimal environment for the growth of BMSCs. Cytokine and gene del ivery could promote BMSCs to differentiate toward chondrocytes. All of them played important roles in the field of cartilage tissue engineering. Conclusion The improvement of three-dimensional scaffolds, the rational use of cytokine, and the enhancement of gene del ivery will promote the development of cl inical cartilage reconstruction.
【Abstract】 Objective To approach the possibil ity of combination of simvastatin and BMSCs transplantation forsteroid-associated osteonecrosis of femoral head. Methods The BMSCs harvested from 24 rabbits were prepared for cell suspension at a concentration of 1 × 107/mL, and combined with gelatin sponge. Seventy New Zealand white rabbits received one intravenous injection of l ipopolysaccharide (10 μg/ kg). After 24 hours, three injections of 20 mg/kg of methylprednisolone were given intramuscularly at a time interval of 24 hours. Forty-eight rabbits diagnosed as having femoral head necrosis by MRI were divided into 4 groups randomly, group A: no treatment; group B: only decompression; group C: decompression and BMSCs transplantation; and group D: simvastatin drench (10 mg/kg.d) decompression and BMSCs transplantation. The general information of animals were recorded; after 4 and 8 weeks of operation, 6 rabbits of each group were chosen randomly to do MRI scan, and femoral heads were harvested to do histopathology and scanning electron microscope examination. Results After 8 weeks, rabbits became more active than before treatment, and walking way became normal gradually in groups C and D. Fourweeks after operation, the MRI low signal region of all groups had no obvious changes, but 8 weeks later, the necrosis signal region of group A magnified while it reduced obviously in group D. Histopathological observation: 4 weeks after operation, diffuse presence of empty lacunae and pyknotic nuclei of osteocytes were found in the trabeculae, and few newborn micrangium could been seen in group A; lots of empty lacunae and a small quantity of newborn micrangium could been found in group B; and large amounts of osteoblats and newborn micrangium were found around the necrosis regions in groups C and D. The positive ratio of empty lacunae and microvessel density in group D were 19.30 ± 1.52 and 7.08 ± 1.09, showing significant difference compared with other groups (P lt; 0.05). After 8 weeks of treatment, the bone trabecula collapsed in many regions in group A; there was fibra callus formation along the decompression channel in group B; few empty lacunae was in the bone trabecular, but the shape of marrow cavity was not normal in group C; and it showed almost normal appearance in group D. The positive ratio of empty lacunae and microvessel density in group D were 11.31 ± 1.28 and 12.37 ± 1.32, showing significant differences compared with other groups (P lt; 0.05), meanwhile, showing significant difference compared with that of 4 weeks after operation(P lt; 0.05). Scanning electron microscope: 8 weeks after operation, the bone trabecula collapsed in many regions, and few osteoblasts could be found on the surface, a great quantity of fat cells cumulated in the bone marrow in group A; cracked bone trabecula could be found occasionally in group B; the density of bone trabecula was lower than the normal in group C; and the shape of the marrow avity and thedensity of bone trabecula were similar to the normal in group D. Conclusion Simvastatin can promote the differentiation of osteocyte and vascular endothel ial cell from MSCs, the combination of simvastatin and marrow stem cells transplantation for the treatment of steroid-associated osteonecrosis of femoral head have good appl ication prospects.
Objective To evaluate the effect of the plasma treated PLGA nerve conduits seeded BMSCs on repairing SD rat sciatic nerve defects. Methods BMSCs were acquired from 30 newborn SD rats. After ampl ified and passaged for 3 times, PLGA nerve conduits were prepared and some of them were treated with plasma. A 1-cm-length sciatic nerve defect wasmade in 30 4-week-old SD rats, then they were randomly divided into 3 groups for three different nerve defects reconstruction methods (n=10). In the experimental group, defect was repaired by plasma treatment and PGLA nerve conduits seeded with BMSCs; in the control group, by normal PLGA nerve conduits seeded with BMSCs; and in the autologous group, by autologous nerve. At 6 weeks after the surgery, the dynamic walking pattern was recorded and the sciatic function index (SFI) was calculated; the electrophysiological test was taken; the gastrocnemius wet weight recovery rate was calculated; and the image analysis of regenerated nerve was made. Results All rats survived after the surgery and l ived to the end of the experiment. At 6 weeks after the surgery, the dynamic walking pattern of the experimental group and autologous group was better than that of the control group. The SFI value of the experimental, control and autologous groups was —51.02 ± 6.54, —58.73 ± 7.87 and —48.73 ± 3.95, respectively, showing statistically significant differences among the experimental group, control group and autologous group (P lt; 0.05). The results of the motor nerve conduction velocity and wave ampl itude showed that there were statistically significant differences between the experimental group and the control group (P lt; 0.05), and between the control group and the autologous group (Plt; 0.01); but no significant difference between the experimental group and autologous group(Pgt; 0.05); The gastrocnemius wet weight recovery rate of the experimental, control and autologous groups was 56.13% ± 4.27%, 43.14% ± 6.52%, 59.47% ± 3.85%, respectively; showing statistically significant differences among experimental group, control group and autologous group (P lt; 0.05). The density, diameter of regenerated nerve fiber as well as neural sheath thickness of the experimental group were all higher than those of the control group (P lt; 0.05) and lower than those of the autologous nerve group (P lt; 0.05); there was significant difference between the control group and the autologous group (P lt; 0.01). Conclusion Plasma treated PLGA nerve conduits seeded with BMSCs can effectively repair sciatic nerve defects and provide a new strategy for the development of tissue engineered nerve to repair the peripheral nerve defects.
Objective To investigate the feasibil ity of inducing canine BMSCs to differentiate into epithel ial cells in vitro with epithel ial cell conditioned medium (ECCM). Methods Five mL BMSCs were obtained from il iac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured. The oral mucosa was harvested and cut into 4 mm × 4 mm after the submucosa tissue was el iminated; ECCM was prepared. BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group. The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting. The cells were identified by immunohistochemical staining through detecting cytokeratin 19 (CK-19) and anti-cytokeratin AE1/AE3 on the21st day of induction. The ultra-structure characteristics were observed under transmission electron microscope. Results The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope. The cell growth curves of two groups presented S type. The cell growth curve of the experimental group was right shifted, showing cell prol iferation inhibition in ECCM. The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, confirming the epithel ial phenotype of the cells; while the result was negative in the control group. The cells were characterized by tight junction under transmission electron microscope. Conclusion The canine ECCM can induce allogenic BMSCs to differentiate into epithel ial cells in vitro.
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
Objective To evaluate sex determining region of the Y (Sry) as a engrafting track of the transplanted BMSCs survival and new bone formation in the osteonecrosis of the femoral head (ONFH) of rabbit. Methods Fortynine 4-5-month-old New Zealand White rabbits were included, weighing 2.0-2.5 kg, 48 females and 1 male. BMSCs of the rabbits were isolated by density gradient separation method, the third passage cells were marked by 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethyl indocarbocyanine perchlorate (DiI) and the concentration of cell suspension was 2.5 × 108/ mL. The animal model of ONFH were establ ished with 48 female rabbits by injecting l iquid nitrogen, and femoral head was not dislocated.The animal model were divided into 3 groups, 16 rabbits in each group. Group A only establ ished animal model as control. Autologous BMSCs (4 μL) marked by DiI was transplanted in the ONFH models of the group B. Allogenic BMSCs (4 μL) marked by DiI was transplanted in ONFH models of the group C. The femoral head were observed by X-ray, HE staining and Masson staining, and the regenerating trabecular volume percentages was determined at 2, 4, 6 and 8 weeks after operation respectively. The examples of the heart, lung, l iver, spleen and kidney were obtained. The transplanted BMSCs were traced by fluorescence microscope, the Sry gene expression was detected by PCR for cells survival. Results All rabbits survived till the end of experiment. The X-ray showed gradual necrosis in the femoral head of group A. HE and Masson staining results indicated that compared with the group A, the recovery condition of the necrotic femoral head in the groups B and C was better. At each time of groups B and C, the regenerating trabecular volume percentages were higher than that of the group A significantly (P lt; 0.01). There was no significant difference between groups B and C (P gt; 0.05). The cells marked by DiI were not founded in the tissues of the heart, lung, l iver, spleen and kidney in groups B and C at each time. PCR showed that the expression of Sry gene were not observed at the heart, lung, l iver, spleen and kidney of three groups at each time. The expression of Sry gene was clearly identified in the femoral head of all 16 rabbits in the group C at each time point. Conclusion Allografting of BMSCs transplanted into the femoral head can survive and induce new bone formation without redistribution.
Objective To investigate a new composite matrix (BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM) bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind l imbs. Methods The human amniotic membrane (HAM) was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats (n=4), then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM (BMSCs-DHAM). The growing state of BMSCs was observed under themicroscopy. Moreover, 40 female rats (8-week-old, weighting 200-220 g) were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). The both stumps were respectively wrapped by BMSCs- DHAM or simple DHAM in groups A and C, and the same dose of BMSCs or physiological sal ine were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindl imbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential (MEP), anterograde biopinylated dextan amine (BDA) tracing, and immunofluorescence of neurofilament protein 200 (NF-200). Results HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score (12.50 ± 1.26) in group A was significantly higher than those of other groups (P lt; 0.05), and the recovery in latency (3.52 ± 2.45) ms and ampl itude (480.68 ± 18.41) μV of MEP was also obviously improved in group A (P lt; 0.05) when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% ± 3.30% under the lesion level in group A was higher than those of other groups (P lt; 0.05), and lots of the regeneration axons (positive NF-200) were found to grow into the spinal cord under the composite matrix in group A. Conclusion The BMSCs-DHAM composite matrix can improve hindl imb motor function to some extent after spinal cord injury. It will be widely appl ied as the matrix material in the future.
Objective To investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
Objective To evaluate the adhesion, prol iferation and osteogenic differentiation of rabbit BMSCs after cultured on freeze-dried demineral ized bone matrix (FDBM) modified with type II cadherin ectodomain (Cad- II). Methods BMSCs isolated from 10 Japanese white rabbits (male and female, 4-week-old, 0.61-0.88 kg) were cultured. The second generation of BMSCs (cell density 1 × 106 /mL) were seeded onto the Cad-II modified allogenic FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro. The densities of seeded cells, the adhesion rate and their ALP activity were measured. The complex was observed through inverted phase contrast microscope and scanning electron microscope to evaluate the interaction between cells and FDBM. Another group of second generation of BMSCs (cell density 5 × 105 /mL) were seeded onto the Cad-II modified FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro too. The ALP activity and osteocalcin immunohistochemical was measured. Results There was no significant difference in cell prol iferation between experimental group and control group. The adhesion rate of cells in the experimental group was 87.41% ± 5.19%, higher than that in the the control group 35.56% ± 1.75% (P lt; 0.01); the densities of seeded cells reached 5.0 × 105, showing significant difference compared with the control group (2.6 × 104, P lt; 0.05). Inverted phase contrast microscope showed that in the experimental group, more cultured BMSCs pasted in the hole and edge of the scaffold than that in the control group. HE staining showed the densities of seeded cells in the experimental group was higher than that in the control group. Scanning electron microscope showed that in the experimental group, a lot of cultured BMSCs adhered, spreaded in the scaffold, in the control group only a few BMSCs unevenly distributed in the scaffold. After 7 days of culture, the cultured BMSCs on modified FDBM expressed higher ALP activity; after 14 days of culture, the ALP activity (29.33 ± 1.53) was higher than that cultured on unmodified FDBM (18.31 ± 1.32), the positive rates of osteocucl in were 83% ± 7% in the experimental group and 56% ± 7% in the control group, showing significant difference (P lt; 0.01). Conclusion Cad-II enhanced cell adhesion to FDBM and promoted BMSCs differentiate to osteoblast, but no obvious effects were observed in cell prol iferation.
Objective To access the possibil ity of CPC as a suitable scaffold for tissue engineering artificial rib by morphologic observation, adhesion experiments and cellar prol iferation experiments. Methods The 5 mm × 5 mm × 5 mm CPCs were prepared and the structure and components of CPC were compared with those of the normal human bone by micro-CT and scanning electron microscope. Bone marrow aspirates were harvested from the young pig and monuclear cells were separated. The first passage cells were collected and re-suspended in the culture media at a density of 6 × 105 cells/mL. There was 150 μL suspension which was incoluated on the CPC, and then cells were recollected and counted 4, 12 and 24 hours after inoculation. MTT was used to examine the growth condition of BMSCs on the surface of CPC. The scanning electron microscope was used to observe the CPC scaffold 7 days after inoculation, and comparison was made with CPC and the normal human bone. Results The adhesion rate of CPC was 28.00% ± 0.98%, 46.70% ± 1.14% and 48.50% ± 1.18%, respectively 4, 12 and 24 hours after compound culture. The prol iferation rate of CPC was 1.103 ± 0.214, 1.557 ± 0.322, 1.920 ± 0.178, 2.564 ± 0.226, 2.951 ± 0.415 and 3.831 ± 0.328, respectively 1, 2, 3, 4, 5 and 6 days after compound culture, with an obvious rising trend. The micro-CT demonstrated that the content of hydroxyapatite of porous phosphate calcium was (1 101.222 8 ± 0.618 4) mg/ ccm while that of the normal human bone was (1 072.552 3 ± 0.744 2) mg/ccm, and the porosity of porous phosphate calcium was 70.26% ± 0.45% while that of the normal human bone was 72.82% ± 0.51%, and there was no significant difference (P gt; 0.05). The experiment of cell prol iferation showed that the cell which was cultivated with porous phosphate calcium prol iferated rapidly. Through the inverted phase contrast microscope, it was found that the cells grew well and there was no dead cell, which indicated that the material had no toxicity. The rate of the cell adhesion to CPC was less than 50%. Conclusion The structure and components of CPC are similar to those of the normal human bone, and BMSCs grow well on the surface of it, so it is asuitable scaffold for tissue engineering artificial rib. However, the cell adhesion abil ity is to be further improved.