Objective To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-β3 and BMP-7 in vitro. Methods The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors’ il iac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were establ ished. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures of hBMSCs were divided into four groups: 10 ng/mL TGF-β3 group (group A), 200 ng/mL BMP-7 group (group B), combination group of TGF-β3 and BMP-7 (group C) and blank group as the control (group D). Histological observation, RT-PCR and RQ-PCR were appl ied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. Results As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviouly changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day (P lt; 0.05). The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day (P lt; 0.05). The expressions of collagen type X only was positive in group A. Conclusion Combination of TGF-β3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.
Objective To review the study progress of mesenchymal stem cells induced to differentiate intervertebral disc cells Methods The recent related literature was reviewed. The theorical and experimental studies were summarized. Results MSCs had the potential of multidirectional differentiation.International experimental studies indicated the potential of MSCs induced to differentiate intervertebral disc cells. Conclusion MSCs induced to differentiate intervertebral disc cells has the fine prospect.
Objective To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to valuate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell prol iferation and phenotype. Methods The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) andco-cultured with notochord cells (group B) (1 ∶ 1), and cell prol iferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. Results The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 μm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 μm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P gt; 0.05). The co-cultured cells (group B) increased in cell prol iferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell prol iferation in group B was more than that in group A after 4-day culture (P lt; 0.05). The cocultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. Conclusion The notochord cells are conducive for the prol iferation and phenotypekeeping of the chondroid cells and may play a key role in preventing degeneration of the disc.
Objective To summarize recent research progress in hydrogel-based growth factors for treatment of intervertebral disc degeneration (IDD). Methods The relevant literature on hydrogel-based growth factors for IDD treatment at home and abroad was extensively reviewed, and their advantages and therapeutic effects in repairing IDD were analyzed and summarized. Results Hydrogels exhibit high hydration, biocompatibility, and biodegradability, enabling targeted delivery and sustained release of growth factors such as growth differentiation factors and transforming growth factors. This facilitates enhanced efficacy in promoting cell proliferation, extracellular matrix synthesis, and reducing inflammatory responses. Consequently, hydrogels demonstrate broad application prospects in the repair of IDD. ConclusionResearch on hydrogel-based growth factors for treating IDD demonstrates advantages such as avoiding disc damage caused by repeated injections and controlling growth factor release concentrations. However, drawbacks include the limited variety of loaded growth factors and the need to verify the long-term stability and biocompatibility of hydrogels. Therefore, further research is required on aspects such as the types of loaded growth factors and the long-term stability and biocompatibility of hydrogels to establish an experimental foundation for their clinical application.
ObjectiveTo investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits.MethodsTwenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L3, 4, L4, 5, and L5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot.ResultsThe comprehensive score of biological response in each group after injection was significantly lower than that before injection (P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups (P<0.05), and there was no significant difference among other groups (P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection (P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection (P<0.05). There was no significant difference between other groups (P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks (P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks (P<0.05), and there was no significant difference between other groups (P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group (P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group (P<0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group (P<0.05). There was no significant difference in the expression of p65 protein between groups (P>0.05).ConclusionZinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.
Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.
Objective To introduce the research of nucleus pulposus cells for treating intervertebral disc degeneration. Methods The original articles in recent years about nucleus pulposus cells for treating intervertebral disc degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. Results Nucleus pulposus cells are not only simply a remnant of embryonic notochordal cells, but have also an important influence on the well-being of the whole disc. The biological treatment strategies aim to regenerate the disc by either trying to improve the micro-enviroment within the disc or to increase the popoulation of the nucleus pulposus, which includes transplanting mesenchymal stem cellsto differentiate into nucleus-l ike cells in the degenerated intervertebral disc. Conclusion Nucleus pulposus cells or ucleus pulposus l ike cells based cell transplantation methods prove to be a promising and real istic approach for the intervertebral disc regeneration.
Objective To investigate the therapeutic effect of BMSCs- chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its cl inical appl ication. Methods Two mill il iter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by 5-BrdU and mixed with chitosan hydrogel to prepare BMSCs- chitosan hydrogel complex. Six rabbitswere selected to establ ish the model of intervertebral disc degeneration and randomized into 3 groups (n=2 per group): control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 μL of autogenous BMSCs- chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 μL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU label ing detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted. Results Cell label ing detection showed that autogenous BMSCs survived and prol iferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O stainning notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was l ighter than that of other two groups, with a indistinct structure.Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the ransplantation and the degeneration group was 223.84 ± 3.93, 221.03 ± 3.53 and 172.50 ± 3.13, respectively, indicating there was no significant difference between the control and the transplantation group (P gt; 0.05), but a significant difference between the control and transplantation groups and the degeneration group (P lt; 0.05). Conclusion The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the cl inical appl ication of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.
ObjectiveTo isolate nucleus pulposus cells (NPCs) from the caudal and lumbar intervertebral disc of rat, and to identify the morphology and to compare the characteristics. MethodsThe whole spine was separated from 8-week-old Sprague Dawley rats under the sterile conditions. NPCs of different segments (lumbar group: L1,2-L6, S1; caudal group: C1,2-C17,18) were cultured by adherent cultivation approach. Cellular morphologic change was noted by HE staining and continuous observation under inverted phase contrast microscope. Besides, the aggrecan and collagen type Ⅱexpression were examined by toluidine blue and immunocytochemistry staining respectively. The total protein contents, senescence level, and the cell viability of passage 1-5 (P1-5) were detected. The growth curves of the P1 cells in lumbar and caudal groups were determined by cell counting kit 8. ResultsThe NPCs were isolated and identified successfully. The adherence time of the primary cells (the cell fusion reached 90%) in lumbar group was significantly longer than that in caudal group in primary generation (P<0.05). HE staining showed that cytoplasm was pink with the blue nucleus. Lumbar disc NPCs were spindle. The larger caudal disc NPCs were polygonal or irregular. Toluidine blue staining showed that the proteoglycan was stained as blue. In the cytoplasm of cells, collagen type Ⅱwas stained as brown surround the blue-black nucleus. The cell viability had no significant difference between lumbar and caudal groups and between different passages in the same group (P>0.05). The caudal disc NPCs reached their logarithmic growth phase after 3 days of culture, while the cells in lumbar segments did after 4-5 days of culture. The cell proliferation in caudal segments was more than that in lumbar segments at 3-9 days (P<0.05). The difference in the total protein contents was not significant between cells at P1-5 in 2 groups (P>0.05), and the caudal disc NPCs had higher protein contents than lumbar disc NPCs (P<0.05). There was no significant difference in cell senescence rate between cells at P1, P2, and P3 in 2 groups (P>0.05), but significant difference was shown in senescence rate between 2 groups in cells at P4 and P5 (P<0.05). ConclusionCaudal disc NPCs have a better status, which is more suitable for experiment as a seed cell than the lumbar disc NPCs in the same generation.
Objective To explore a practical method of culturing discs organ system by observing the changes of the nucleus pulposus after the whole intervertebral discs (including cartilage end-plate, nucleus pulposus, and annulus fibrous)were cultivated. Methods A total of 335 intervertebral discs were taken out completely from 60 healthy SD rats (about150 g) aged 5-6 weeks of clear grade and rinsed by high osmotic sal ine solution containing heparin, then put to the culture plate after being divided into 5 groups randomly. The whole intervertebral discs were cultured with high osmotic (410 mOsmol/ kg) culture medium and changed the medium once every day, then the cell viabil ity (n=15), HE staining (n=15), Safranin O staining (n=15), and immunohistochemistry staining (n=2) were observed at 0, 3, 7, 14, and 21 days; RT-PCR result (n=5) was observed at 0, 3, 7, and 14 days. Results The cell viabil ity was not changed significantly within 14 days (P gt; 0.05) and was significantly lower at 21 days than at other time points (P lt; 0.01). The immunohistochemistry staining results for collagen type II were positive in nucleus pulposus cells at every time point. HE staining showed that the tissue integrity and morphology of the whole intervertebral discs were not changed within 14 days. Safranin O staining showed no significant difference in the matrix grey scale within 14 days (P gt; 0.05) and significant differences between 21 days and 0-14 days (P lt; 0.05). RT-PCR results showed that the mRNA expression of collagen type I increased with time, but the expressions of collagen type II, aggrecan, and decorin decreased, showing significant differences in the mRNA expressions of the matrix protein at each time point (P lt; 0.05). Conclusion High osmotic sal ine solution containing heparin could be used to cultivate the whole intervertebral discs, it is an ideal model for futher studies on physiology and pathology of intervertebral discs.