OBJECTIVE: To investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature. METHODS: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later. RESULTS: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked. CONCLUSION: The cryopreservation agent in group A can protect TET in deep low temperature.
Objective As one of the adult stem cells, adi pose-derived stem cells (ADSCs) have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs invitro. Methods The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cool ing with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in l iquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkal ine phosphatase (ALP) staining and Al izarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured. Results The survival percentage of the cryopreserved cells was 90.44% ± 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Al izarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group (P gt; 0.05). Conclusion Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.
Objective To investigate the effect of cryopreservation (CP) on the expression of connective tissue growth factor (CTGF) in the renal tubular epithel ial cells. Methods A total of 40 male Wistar rats (weighing 230-250 g) were used in this study. En bloc removal with in situ cooling both kidneys and hypertonic citrate adenine preservation solution were adopted. The rat kidney was be preserved 0, 12, 24, 36 and 48 hours at 0-4℃ (n=8), respectively. The expression of CTGF of renal tubularepithel ial cells was detected by using immunohistochemistry and in situ hybridization analysis. Results The expression of CTGF was less in CP 0 hour group and CP 12 hours group, the positive unit (PU) values of CTGF protein were 5.91 ± 2.30 and 5.57 ± 2.40 (P gt; 0.05), respectively, and the PU values of CTGF mRNA were 6.24 ± 2.79 and 6.51 ± 2.43 (P gt; 0.05), respectively. The PU values of CTGF protein increased at CP 24 hours group (10.25 ± 2.92), CP 36 hours group (14.31 ± 2.83) and CP 48 hours group (18.11 ± 3.94, P lt; 0.05), respectively, and the PU values of CTGF mRNA increased at CP 24 hours group (15.24 ± 3.95), CP 36 hours group (19.20 ± 4.73) and CP 48 hours group (23.09 ± 4.40, P lt; 0.05), respectively; showing significant differences when compared with CP 0 hour group and CP 12 hours group (P lt; 0.05). Conclusion CTGF expression may increase with severe cold ischemia injury, and might play an important role in regeneration and repair of renal tubular epithel ial cell injury.
Objective To investigate the effect of tissue engineered bone with cryopreservation on healing of bone defects and to explore feasibility of cryopreservation for tissue engineered bone. Methods Tissue engineeredbones were constructed with osteoblasts being seeded onto bio-derived materials made from freshhuman bones,and they were preserved at 4℃ and －196℃ for 3 months and 6 monthsrespectively.They were applied to repair segmental bone defects of rabbit’s radius while the tissue engineered bone without cryopreservation and bio-derived materials were brought into control groups.The experiment was divided into groups A3,A6,B3,B6,C and D(group A3:tissue engineered bones were preserved at 4℃ for 3 months; group A6:tissue engineered bones were preserved at 4℃ for 6 months;group B3:tissue engineered bones were preserved at －196℃ for 3 months; group B6:tissue engineered bones were preserved at －196℃ for 6 months; group C: tissueengineered bones without cryopreservation; group D: bio-derived materials). Macroscopical and histologial examination were done at the 2nd,4th,6th,12th weeks, X-ray examination was done at the 6th,12th weeks and biomechanics were determined at 12th weeks after operation respectively. Results Macroscopical observation showed no significant differences among group A3, A6, B3, B6 and C, but less new bone formation and more obvious boundary in group D were observed. Histological observation showed more collagen and new bone around the edge of implant of group A3, A6, B3, B6 and C than group D, and histological evaluation showed significant differences between group D and other groups(P＜0.05). Radiographic observation showed no absorbability of the implant cortex and less new bone formation in group D, but the unity between implant and host bone, medullary cavity reopened, disappearance of fracture line and fine bone modelling were observed in other groups at 12 weeks after operation. Biomechanics between group D and other groups showed significant differences(P＜0.05). Conclusion Cryopreservation (4℃ and -196℃) were capable of preserving tissue engineered bone for long time, and tissue engineered bone withcryopreservation has significant effect on healing of bone defects. The methods f it clinical application.
Objective To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons.Methods In the 4thgeneration of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342(Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguishthem. The seeding cells were collected to make them to be the cell suspension of suitable concentration(6.0×105 cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and didnot cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to noncryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreservedcell to non-cryopreserved cell and the cell survival rates. We compared the cll survival rates between immediate flow cytometry and that 2 hours after fluorescence staining. Results The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r=0.970，Plt;0.05), image analysis study also showed the correlation was significant (r=0.982，Plt;0.05).The cell survival rate decreased by use of flow cytometry twohours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (Pgt;0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly.Conclusion Flow cytometry and fluorescence image afterPI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservationof tissue engineered tendons.
In order to repair cartilage defect in joint with transplantation of cryopreserved homologous embryonic periosteum, 30 rabbits were used and divided into two groups. A 4 mm x 7 mm whole thickness cartilage defect was made in the patellar groove of femur of each rabbit. The homologous embryonic rabbit skull periosteum (ERSP), preserved in two-step freezing schedule, was transplanted onto the cartilage defect of joints of one group and autogenous periosteal graft was done in the joint defect of the other group. The knees were not immobilized, following operation and 16 weeks later, the newly formed tissue in the defects were assessed by gross observation, histochemical examination and biochemical analysis. The results showed that new hyaline-like cartilage was formed in the cryopreserved ERSP grafted knee, and had no significant difference from that of the knee receiving autogenous periosteal graft, but had significant difference from that of the fresh ERSP grafted knee and the non-grafted knee. Furthermore, the new hyaline-like cartilage had the biochemical characteristics of a fibrous cartilage. The conclusion was that this method might be feasible to repair articular cartilage defects.
Objective To investigate the effects of different temperatures on the system of in vitro physiological environment fostering limbs. Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups (n=6) according to different temperatures: limbs were placed in in vitro physiological environment foster-ing limbs at 26℃ (group A), 4℃ (group B), 10℃ (group C), and 18℃(group D). After 12 hours of perfusion, the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope. The mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected by real-time fluorescent quantitative PCR (RT-qPCR). Results Histological results showed that the skeletal muscle exhibited mild edema, integrity of the sarcolemma, and occasional perivascular inflammatory cell infiltration in groups B, C, and D, meanwhile, the cells of group C had normal morphology; however, muscle fibers degenerated, muscle cells were seriously damaged, a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A. Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder, and many focal solubility necrosis occurred in group A; the muscle fibers arranged in order relatively and sarcolemma was still intact, with mild swelling and flocculent degenerative mitochondria in group B; a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C; and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D. RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B, C, and D (P lt; 0.05); the expressions were significantly lower in groups B and C than in group D, and in group C than in group B (P lt; 0.05). Conclusion In the system of in vitro physiological environment fostering limbs, temperature plays an important role in the preservation of amputated limbs. It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.
Objective To observe the long-term effectiveness of tendon allograft to repair tendon defect. Methods Between October 1996 and September 1999, 24 patients with tendon defect were treated with tendon allograft which was cultured with deoxyguanosine and preserved at low-temperature or ultra-deep-low-temperature. There were 19 males and 5 females, aged from 12 to 46 years with an average of 25.9 years. These patients included 7 cases of total extensor tendon defect of 2nd-5th fingers, 7 cases of index finger extensor tendon defect, 3 cases of deep flexor tendon defect of 2nd- 5th fingers, 1 case of ring finger deep flexor tendon defect, 3 cases of long extensor tendon defect of 2nd-5th toes, 2 cases of long extensor hallucis tendon defect, and 1 case of shoulder adduction missing. The sizes of tendon defect ranged from 5 to 15 cm. The mean time from injury to operation was 1.3 months (range, 2 hours to 3 months). Results Incisions healed by first intention. No deep infection, infectious diseases, and obvious immune rejection occurred. All patients were followed up from 10 to 12 years with an average of 10.8 years. When compared with contralateral sides, at 10 years of follow-up, 1 patient lost 6-10° flexion function; after 10.6 years, flexion tendon releasing was performed; allografted tendon had normal color and elasticity with decreased diameter and with mild and moderate adherence; and after releasing, function was improved. According to Hand Surgery Association assessment standard, the results were excellent in 12 cases, good in 6, and poor in 6; the excellent and good rate was 75%. Conclusion Tendon allograft which is cultured with deoxyguanosine and preserved at low-temperature or ultra-deep-low-temperature is safe to use in cl inical, which has good long-term effectiveness in treating tendon defect.
Objective o study the feasibility of homologous vascularized nerve transplantation after ultra deep cryopreservation. Methods Vascularized sciatic nerve from 12 female dogs was transplanted after ultra deep cryopreservation. Fortyeight male dogs were divided into 4 groups: ultra deep cryopreservation homologous vascularized nerve (group A), ultra deep cryopreservation homologous nerve (group B), fresh homologous vascularized nerve (group C), and fresh autologous vascularized nerve (group D). The gross appearance, patency rate of arteryand morphological transplanted nerve were observed 1, 4 and 12 weeks after transplantation respectively. Immunological analysis was performed using IL 2 assay and T lymphocyte subpopulations assay after 4 weeks. Image pattern analysis andelectromyogram were observed after 12 weeks. Results In groups A and D, no toe ulcer occurred, the atrophy of later limb and the sense of pain from skin of calf were restore significantly in the postoperative 12th week. In groups B and C, toe ulcer occurred, the atrophy of later limb and the sense of pain from skin of calf were not restored significantly in the postoperative 12thweek. The vessel patency rate of groups A and D was 83.3%, which was significantly higher than that of group C (50%,Plt;0.05). The changes of IL2 and Th, Ts in group C were significantly higher than that in groups A,B,D(Plt;0.01). There were increased vessel and regenerated nerve in transplanted nerve under optical microscope and image pattern analysis in groups A and D. There were shorter latent period of motor evoked potential, greater amplitude of action potenlial and faster motor nerve conducting velocity in groups A and D after 12 weeks. Conclusion The antigenicity of the homologous never and vessel may be reduced significantly by being frozen, and cryopreserved vascularized nerve can transferred successfully without the use of immunosuppressive agents. Vascularized nerve may restore good significantly for the thick nerve.
ObjectiveTo explore the effects of cryopreservation on the cell survival rate, cell viability, early apoptosis, migration ability, and tendon-related marker expression of tendon-derived stem cells (TDSCs) in rat patellar tendons.MethodsThe patellar tendon tissues were harvested from 12 4-month-old male Sprague Dawley rats; 12 patellar tendon tissues from 6 rats were cryopreserved (the experimental group), and the other 12 patellar tendon tissues were not treated (the control group). The patellar tendons were digested with 0.3% type I collagenase to obtain nucleated cells. The survival rate of nucleated cells was detected by trypan blue exclusion assay, and colony-forming ability by crystal violet staining. TDSCs were isolated and cultured to passage 3 (P3). The cell viability of TDSCs was detected by Alamar Blue method, the early apoptosis by Annexin V-FITC/PI assay, the cell migration ability by Transwell method, and the mRNA expressions of tendon-related markers [collagen type I (Col1α1), scleraxis (Scx), and tenomodulin (Tnmd)] by real-time quantitative PCR.ResultsThe survival rate of nucleated cells was 91.00%±3.63% in the control group, and was 61.65%±4.76% in the experimental group, showing significant difference (t=12.010, P=0.000). The formation of the primary nucleated cell clones was observed in 2 groups. At 12 days, the number of colonies forming of the experimental group [(8.41±0.33)/1 000 nucleated cells] was significantly lower than that of the control group [(15.19±0.47)/1 000 nucleated cells] (t=28.910, P=0.000). The percentage of TDSCs in the active nucleated cells in the experimental group (1.37%±0.09%) was significantly lower than that in the control group (1.67%±0.10%) (t=5.508, P=0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance (A) value between 2 groups at each time point (P>0.05). The early apoptotic rate of TDSCs was 1.67%±0.06% in the experimental group and was 1.63%±0.06% in the control group, showing no significant difference (t=0.707, P=0.519). Under microscope, TDSCs adhered to the lower chamber of the Transwell chamber; the number of cells was 445.00±9.70 in the experimental group and was 451.50±12.66 in the control group, showing no significant difference (t=0.998, P=0.342). The relative mRNA expressions of Col1α1, Scx, and Tnmd were 3.498±0.065, 0.062±0.002, and (4.211±0.211)×10–5 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10–5 in the con-trol group, showing no significant difference (t=0.013, P=0.991; t=0.042, P=0.969; t=0.653, P=0.549).ConclusionThe survival rate of nucleated cells in cryopreserved rat tendon tissues is lower, but a large number of active TDSCs, and its cell viability, early apoptosis rate, migration ability in vitro, and cell tenogenic differentiation ability are remained.