Stem cell therapy for osteonecrosis of femoral head: Opportunities and challenges.

Osteonecrosis of the femoral head (ONFH) is a progressive disease with a complex etiology and unclear pathogenesis, resulting in severe hip pain and dysfunction mainly observed in young patients. Although total hip arthroplasty (THA) is the most effective treatment for patients with ONFH in the terminal stage, the results of THA in young patients or active populations are often not favorable, with some complications related to the prosthesis. With the development of biotechnology, an increasing number of studies pay attention to use of stem cells for the treatment of ONFH. Stem cells are characterized by the ability to self-renew and differentiate into multiple cell types, including differentiation into osteoblasts and endothelial cells to mediate bone repair and angiogenesis. Furthermore, stem cells can offer growth factors to promote blood supply in the necrotic regions by paracrine effects. Therefore, stem cell therapy has become one of the hip-preserving alternatives for ONFH. This review summarized the current trends in stem cell therapy for ONFH, from clinical applications to related basic research, and showed that an increasing number of studies have confirmed the effectiveness of stem cell therapy in ONFH. However, many unsolved problems and challenges in practical applications of stem cell therapy still exist, such as patient selection, standardized procedures, safety assessment, and the fate of transplanted cells in the body. Additional studies are required to find ideal cell sources, appropriate transplantation methods, and the optimal number of cells for transplantation.

Tendon healing in vivo: effect of mannose-6-phosphate on flexor tendon adhesion formation.

The purpose of this study was to investigate the preventive effect of mannose-6-phosphate on flexor tendon adhesion formation. From a total of 84 adult New Zealand White rabbits, 36 were randomly divided into 2 groups, the normal saline group and the mannose-6-phosphate group, after anastomosis of the flexor tendons. Tendons were harvested at 4 weeks, and biomechanics testing was conducted. The other 48 rabbits were randomly divided into 2 groups, the normal saline group and the mannose-6-phosphate group, after anastomosis of the flexor tendons, and tendons were harvested at 7, 14, 28, and 56 days and analyzed by in situ hybridization to determine the mRNA expression of transforming growth factor (TGF)-ß1 and collagen I. The results of biomechanics testing indicated that mannose-6-phosphate can effectively prevent flexor tendon adhesion formation after anastomonsis. The in situ hybridization examination revealed that TGF-ß1 and collagen I mRNA expression in the mannose-6-phosphate group was lower than that in the normal saline group at each time point. Mannose-6-phosphate can effectively inhibit the function of TGF-ß1 and prevent adhesion formation after flexor tendon injury.

A prospective randomized comparison of irradiated and non-irradiated hamstring tendon allograft for ACL reconstruction.

PURPOSE: To analyze the outcome of arthroscopic anterior cruciate ligament reconstruction with irradiated versus non-irradiated hamstring tendon allograft. METHODS: All hamstring tendon allografts were obtained from a single certified tissue bank, and the irradiated allografts were sterilized with 2.5 Mrad of irradiation prior to distribution. A total of 78 patients who met the inclusion and exclusion criteria of the study were prospectively randomized consecutively into two groups (Non-ir-Allo vs. Ir-Allo). All the operations were done by the same senior surgeon. Before surgery and at follow-up, patients were evaluated by the same observer according to clinical evaluations including the Lachman test, ADT, pivot shift test, varus/valgus stress test, the instrumented KT-2000 arthrometer testing, vertical jump test, one-leg hop test, ROM of knee, Cincinnati knee score, IKDC Subjective Knee Form, Tegner activity score, modified Lysholm knee scoring scale, and the standard knee ligament evaluation form of the IKDC. RESULTS: Of these patients, 69 (Non-ir-Allo 38, Ir-Allo 31) were available for full evaluation. When compared the Ir-Allo group with Non-ir-Allo group at the final follow-up by Lachman test, ADT, pivot shift test, and KT-2000 arthrometer testing, statistically significant differences were found (P < 0.05). Most importantly, 84% of patients in the Non-ir-Auto group and just only 32% in the Ir-Allo group had a side-to-side difference of less than 3 mm according to KT-2000. The anterior and rotational stability decreased significantly in the Ir-Allo group. According to the overall IKDC, functional, subjective evaluations, and activity level testing, no statistically significant differences were found between the two groups (n.s.). As to the osteoarthritis (OA) rate, for the Non-ir-Allo group, there was no significant difference (n.s.) in development of OA between the operated knee and contralateral knee at the final follow-up. While for the Ir-Allo group, significant difference (P < 0.05) was found in development of OA between the operated knee and contralateral knee. There was statistical difference (P < 0.05) between the Non-ir-Allo and Ir-Allo groups when comparing the development of OA of the operated knees at the final follow-up. CONCLUSION: There was a significant difference in knee stability between the two groups (in favor of Non-ir-Allo), but no differences in functional scores should be pointed out clearly. LEVEL OF EVIDENCE: I.

Arthroscopic anterior cruciate ligament reconstruction with at least 2.5 years' follow-up comparing hamstring tendon autograft and irradiated allograft.

PURPOSE: To compare the clinical outcomes of arthroscopic anterior cruciate ligament (ACL) reconstruction with hamstring tendon autograft versus irradiated allograft. METHODS: All irradiated hamstring tendon allografts (gracilis and semitendinosus), which were sterilized with 2.5 Mrad of irradiation before distribution, were obtained from a single certified tissue bank. A total of 78 patients undergoing arthroscopic ACL reconstruction were prospectively randomized consecutively into 1 of 2 groups: autograft and irradiated allograft. The same surgical technique was used in all operations, which were performed by the same senior surgeon. Before surgery and at a mean of 42.2 months of follow-up, patients were evaluated by the same observer according to objective and subjective clinical evaluations. RESULTS: Of the patients, 67 (36 in autograft group and 31 in irradiated allograft group) were available for full evaluation. When the irradiated allograft group was compared with the autograft group at the final follow-up by the Lachman test, anterior drawer test, pivot-shift test, and KT-2000 arthrometer (MEDmetric, San Diego, CA) assessment, statistically significant differences were found (P = .00011, P = .00016, P = .008, and P = .00021, respectively). Most importantly, 86.1% of patients in the autograft group and only 32.3% in the irradiated allograft group had a side-to-side difference of less than 3 mm according to KT-2000 assessment. The rate of laxity (side-to-side difference >5 mm) with irradiated allograft (32.3%) was higher than that with autograft (8.3%). The anterior and rotational stabilities decreased significantly in the irradiated allograft group. According to the overall International Knee Documentation Committee rating, functional and subjective evaluations, and activity level testing, no statistically significant differences were found between the 2 groups. However, patients in the irradiated allograft group had a shorter operative time and a longer duration of postoperative fever. When the patients had a fever, the laboratory examination findings of all patients were almost normal (white blood cell count, normal; erythrocyte sedimentation rate, 8 to 20 mm/h; and C-reactive protein level, 4 to 11 mg/L). CONCLUSIONS: The clinical outcome of ACL reconstruction with hamstring tendon autograft was satisfactory, whereas the difference in instrumented laxity between the 2 groups was significant and the difference in functional test results was not significant. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Arthroscopic reconstruction of the anterior cruciate ligament with hamstring tendon autograft and fresh-frozen allograft: a prospective, randomized controlled study.

BACKGROUND: Most studies of allograft versus autograft for anterior cruciate ligament reconstruction have been of bone-patellar tendon-bone; outcome reports evaluating anterior cruciate ligament reconstruction with hamstring tendon autograft versus allograft are rare. PURPOSE: This study was undertaken to compare the clinical outcome of arthroscopic anterior cruciate ligament reconstruction with hamstring tendon autograft versus allograft. STUDY DESIGN: Randomized controlled trial; Level of evidence, 2. METHODS: Between 2000 and 2004, 208 patients who met the inclusion and exclusion criteria of the study were prospectively randomized into autograft (n = 104) or allograft (n = 104) groups. All hamstring tendon allografts were fresh-frozen and obtained from a single certified tissue bank. All the operations were done by the same surgeon with the same surgical technique. Femoral and tibial fixation was by means of an EndoButton and a bioabsorbable interference screw augmented with a staple, respectively. Patients were evaluated preoperatively and postoperatively. Evaluations included detailed history, physical examination, functional knee ligament testing, KT-2000 arthrometer testing, Harner vertical jump and Daniel 1-legged hop tests, Lysholm score, Tegner score, the International Knee Documentation Committee (IKDC) standard evaluation form, Cincinnati knee score, and radiographs. RESULTS: Of these patients, 186 (autograft, n = 91; allograft, n = 95) were available for full evaluation. Demographic data were comparable between groups. The mean follow-up was 7.8 years for both groups. There were no statistically significant differences according to the evaluations of the outcome aforementioned between the 2 groups except that patients in the allograft group had a shorter operation time than the autograft group. Seven patients (7.7%) in the autograft group and 8 (8.4%) in the allograft group had a side-to-side difference >5 mm. Eighty-five patients (93.4%) in the autograft group and 86 (90.5%) in the allograft group were normal or nearly normal according to the overall IKDC. According to the subjective IKDC, the average scores were 89 and 90 points, respectively, for the autograft and allograft groups. The mean Lysholm and Tegner scores were 89 points and 7.7 points, respectively, for the autograft group and 90 points and 7.6 points, respectively, for the allograft group. For the Cincinnati knee score, the average scores were 90 and 91 points, respectively, for the autograft and allograft groups. CONCLUSION: Both groups of patients achieved almost the same satisfactory outcome at an average of 7.8 years of follow-up. Fresh-frozen hamstring tendon allograft is a reasonable alternative choice to autograft for anterior cruciate ligament reconstruction.

Inhibition effect of mannose-6-phosphate on expression of transforming growth factor Beta receptor in flexor tendon cells.

Transforming growth factor beta (TGF-ß) has an important role in tendon healing and adhesion formation. Inhibiting TGF-ß and its receptor expression may prevent adhesions after tendon open. The goal of this study was to examine the effects of mannose-6-phosphate, a natural inhibitor of TGF-ß, on TGF-ß and its receptor production in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes of rabbit flexor tendons. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. The cells were divided into 2 groups at random: an experiment group supplemented with mannose-6-phosphate and a control group without mannose-6-phosphate. The expression of TGF-ß and TGF-ß receptor was quantified with enzyme-linked immunosorbent assay. The luciferase assay measured TGF-ß bioactivity. Transforming growth factor beta expression in the experimental group was not decreased compared with the control group, with no significant difference (P>.05) Transforming growth factor beta receptor expression in the experiment group was significantly lower than that in control group (P<.05). Mannose-6-phosphate significantly decreased the expression of TGF-ß receptor and TGF-ß bioactivity. Modulation of mannose-6-phosphate levels may provide a means of modulating the effects of TGF-ß on adhesion formation in flexor tendon wound healing.

Tendon healing in vivo and in vitro: neutralizing antibody to TGF-ß improves range of motion after flexor tendon repair.

Adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath results in a decreased postoperative range of motion (ROM) in the hand. Transforming growth factor-beta (TGF-ß) is a key cytokine in the pathogenesis of tissue fibrosis. In this study, the effects of TGF-ß1 neutralizing antibody were investigated in vitro and in vivo. In the in vitro investigation, primary cell cultures from rabbit flexor tendon sheath, epitenon, and endotenon were established and each was supplemented with TGF-ß along with increasing doses of TGF-ß1 neutralizing antibody. Collagen I production was measured with enzyme-linked immunosorbent assay. In the in vivo study, rabbit zone-II flexor tendons were transected and then immediately repaired. Transforming growth factor-ß1 neutralizing antibody or phosphate-buffered saline solution (control) was added to the repair sites, and the forepaws were tested for ROM and repair strength at 8 weeks postoperatively. Transforming growth factor-ß1 neutralizing antibody reduced TGF-ß upregulated collagen production. Intraoperative application of TGF-ß1 neutralizing antibody significantly improved the ROM of the operatively treated digits. The effect on breaking strength of the tendon repair was inconclusive.

Inhibition of TGF-ß-induced collagen production in rabbit flexor tendon with mannose-6-phosphate in vitro.

The flexor tendon affects postoperative range of motion in the hand. Transforming growth factor-beta (TGF-ß) is a key cytokine in the adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath. The purpose of this study was to examine the inhibition of TGF-ß-induced collagen-I production in rabbit flexor tendons with mannose-6-phosphate in vitro. Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes from rabbit flexor tendons were isolated and each was supplemented with TGF-ß along with increasing doses of mannose-6-phosphate. The enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR) measured collagen-I production. The luciferase assay measured TGF-ß bioactivity. Results were compared with TGF-ß alone and unsupplemented controls. TGF-ß-induced collagen-I production was downregulated significantly with the addition of mannose-6-phosphate in a dose-dependent manner in all 3 cells cultures. The mannose-6-phosphate also reduced TGF-ß bioactivity. The study shows that mannose-6-phosphate was effective in TGF-ß inhibition in cultured flexor tendon cells. The findings presented here encourage further experiments that use the agents to modulate TGF-ß levels and reduce adhesion formation after flexor tendon repair.

[Effects of melatonin on expression of bone morphogenetic protein 2 and interleukin 1beta in articular cartilage of rat with osteoarthritis].

OBJECTIVE: Melatonin (MLT) can increase the expression of cartilage-derived growth factor and stimulate the synthesis of cartilage matrix. To investigate the prevention and treatment effects of MLT on damaged cartilage through observing the expressions of bone morphogenetic protein 2 (BMP-2) and interleukin 1beta (IL-1beta) in articular cartilage of the rats with osteoarthritis (OA). METHODS: Forty SPF 4-week-old male SD rats (weighing 120-150 g) were randomly divided into 4 groups (n=10): normal control group (group A), OA group (group B), OA/pinealectomy group (group C), and OA/pinealectomy/MLT group (group D). The rats of group A served as a control without treatment. The rats of groups B, C, and D underwent left knee joint injection of 0.2 mL 4% papain solution 1 time every other day for 2 weeks for establishing OA model. Two weeks after papain injection, the rats of groups C and D were exposed to continuous light for 24 hours (intensity of illumination: 500 lx) for creating pinealectomy models. And at the next day after pinealectomy model establishing, the rats of group D were treated with intra-articular injections of 0.2 mL 20 mg/mL MLT solution 4 times a week for 4 weeks. At 1 week after last MLT injection, the venous blood samples were taken in groups A, B, and C to test the level of serum MLT by ELISA, respectively, and then the specimens of left cartilage of femoral condyle were harvested for macroscopic, histological, and immunohistochemical examinations in 4 groups. RESULTS: The OA and pinealectomy models of rats were successfully established, and all rats survived. There were significant differences in the serum MLT level among groups A, B, and C, and among different time points at the same group (P < 0.05). In group A, articular cartilage surface was smooth and elastic, and chondrocytes arranged regularly. In groups B and C, articular cartilage surface was rough, cartilage defects and subchondral bone exposure were observed in some areas, and chondrocytes arranged irregularly. In group D, cartilage surface was more smooth than that in groups B and C, and the degrees of cartilage defect and subchondral bone exposure decreased with regular arrangement of chondrocytes. There were significant differences in Mankin scores and integral absorbance values among 4 groups (P < 0.05). CONCLUSION: Exposure to continuous light can accelerate degeneration process of articular cartilage of OA rats. Injections of 0.2 mL MLT solution (20 mg/mL) by intra-articular for 4 weeks can inhibit the progress of cartilage defects. Upregulation of anabolic factor of BMP-2 as well as down-regulation of catabolic factors of IL-1beta is associated with cartilage repair in the pathological features of OA.

Effects of antisense transforming growth factor-beta1 gene transfer on the biological activities of tendon sheath fibroblasts.

Recent studies have shown the importance of transforming growth factor-beta (TGF-beta) in flexor tendon wound healing. Decreased adhesion formation and increased range of motion after the administration of TGF-beta antibodies after tendon repair have been shown. But TGF-beta antibodies have a short biologic half-life, and continuous supplementation of exogenous TGF-beta antibodies is not practical. Transfer of growth factor genes to tenocytes provides an alternative to protein therapeutics, and a gene therapy approach will prolong the availability of therapeutic proteins.We investigated the biological activities effects of rabbit tendon sheath fibroblasts transfected by antisense TGF-beta1 gene. Tendon sheath fibroblasts were isolated from New Zealand white rabbits and transfected by antisense TGF-beta1 gene with Lipofectin (Invitrogen, Carlsbad, California). Reverse transcription polymerase chain reaction was used to measure collagen I, collagen III, and TGF-beta1 expression, and Western blot was used to measure collagen protein I expression in tendon sheath fibroblasts after being transfected by antisense TGF-beta1 gene. Reverse transcription polymerase chain reaction displayed that tendon sheath fibroblasts transfected with antisense TGF-beta1 gene showed marked decrease collagen I, collagen III, and TGF-beta1 mRNA expression. Western blot showed that tendon sheath fibroblasts transfected with antisense TGF-beta1 gene showed marked decrease expression of collagen I protein, and there was significant difference compared with the untransfected and empty transfected groups (P<.01). Tendon sheath fibroblasts can transfect with antisense TGF-beta1 gene successfully and can decrease production of collagen I, collagen III, and TGF-beta1, which were factors of tendon adhere formation.

[A prospective study on anterior cruciate ligament reconstruction with patellar tendon autograft versus gamma irradiated allograft].

OBJECTIVE: To analyze the stability and clinical outcomes of arthroscopic anterior cruciate ligament (ACL) reconstruction with gamma irradiated patellar tendon allograft compared with autograft. METHODS: From January 2004 to October 2007, 69 patients undergoing arthroscopic ACL reconstruction were prospectively randomized consecutively into two groups: group A (autograft, n=36) and group B (gamma irradiated allograft, n=33). In group A, there were 30 males and 6 females with an average age of 30.1 years, including 30 cases of simple ACL rupture and 6 cases of ACL rupture with medial accessory ligament injury; ACL rupture was caused by sports in 28 cases, by traffic accident in 5 cases, and by others in 3 cases; and the time from injury to operation was 1.4 months on average. In group B, there were 26 males and 7 females with an average age of 32.5 years, including 27 cases of simple ACL rupture and 6 cases of ACL rupture with medial accessory ligament injury; ACL rupture was caused by sports in 27 cases, by traffic accident in 4 cases, and by others in 2 cases; and the time from injury to operation was 1.5 months on average. There were no significant differences in general data between two groups (P > 0.05). The same arthroscopic technique was used in all ACL reconstructions done by the same surgeon. The clinical outcome was evaluated and compared by general conditions, pivot shift test, Lachman test, KT-2000 arthrometer testing, Daniel's one-leg hop test, International Knee Documental Committee (IKDC) scoring, Lysholm knee scoring scale, and Tegner activity score. RESULTS: All patients were followed up for 39.5 months (group A) and 37.6 months (group B). In group A, patella fracture occurred in 1 case and anterior knee pain in 2 cases postoperatively. No complication occurred in group B. The hospitalization times in groups A and B were (15.6 +/- 2.4) days and (15.5 +/- 1.5) days, respectively, showing no significant difference (P > 0.05). The operation time of group A was longer than that of group B and the fever time of group A was shorter than that of group B, showing significant differences (P < 0.05). At the final follow-up, there were significant differences (P < 0.05) in Lachman test and the pivot shift test between two groups, between pre- and post-operation; there. were no significant differences (P > 0.05) in Daniel's one-leg hop test, the IKDC, Lysholm, and Tegner activity scores between two groups, however, there was a decreased trend in the functional and activity levels in group B. And there was significant difference between pre- and post-operation (P < 0.05). At the final follow-up, the differences between normal side and affected side were (2.4 +/- 0.6) mm in group A and (5.5 +/- 3.6) mm in group B, showing significant difference (P < 0.05). There was significant difference in tibial advancement between pre- and post-operation (P < 0.05). CONCLUSION: The functional and activity level of the knee after ACL reconstruction with autograft and gamma irradiated patellar tendon allograft were similar, but anterior and rotational stability of the involved knee decreases significantly in the group with gamma irradiated patellar tendon allograft.

[Effects of mannose-6-phosphate on transforming growth factor beta and transforming growth factor beta receptor expression of flexor tendon cells].

OBJECTIVE: By culturing tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes of rabbits' tendon in vitro, to study the effects of mannose-6-phosphate on transforming growth factor beta (TGF-beta) peptide and receptor expression, and to provide the experimental basis for preventing the tendon healing adhesion by mannose-6-phosphate. METHODS: Eight adult New Zealand white rabbits, regardless of their gender and weighing 4.0-4.5 kg, were selected. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All 3 cells were divided into 2 groups at random after cells were adjusted to a concentration of 4 x 10(4) per well and 1 x 10(4)/mL. The first was the control group without supplementation. The experimental group was supplemented with mannose-6-phosphate. The expressions of TGF-beta and TGF-beta receptor were quantified with enzyme-linked immunosorbent assay. The expression of TGF-beta1 mRNA was also assessed with in situ hybridization and the expression of TGF-beta1 was assessed with immunohistochemistry. RESULTS: The expressions of TGF-beta and TGF-beta receptor in experimental group were significantly lower than that in control group (P < 0.05). The expression levels of TGF-beta1 and TGF-beta2 decreased in descending order of tendon sheath fibroblasts (36.1%, 37.9%), epitenon tenocytes (31.0%, 32.1%), and endotenon tenocytes (31.2%, 27.0%). The expression levels of TGF-beta3 decreased in descending order of endotenon tenocytes (42.5%), tendon sheath fibroblasts (41.2%), and epitenon tenocytes (33.3%). The expression levels of TGF-beta receptor 1 and TGF-beta receptor 2 decreased in descending order of epitenon tenocytes (29.9%, 26.2%), endotenon tenocytes (27.8%, 23.5%), and tendon sheath fibroblasts (23.1%, 20.0%). The expression levels of TGF-beta receptor 3 decreased in descending order of endotenon tenocytes (26.1%), epitenon tenocytes (19.2%), and tendon sheath fibroblasts (15.8%). In experimental group, the positive expression of TGF-beta1 mRNA and the expression level of intracellular TGF-beta1 mRNA in all 3 tendon cells were significantly lower than those in the control group (P < 0.05). Immunohistochemical staining showed the expressions of TGF-beta1 in all 3 tendon cells were significantly lower in the experimental group than in the control group. CONCLUSION: Mannose-6-phosphate can significantly decrease the expressions of TGF-beta peptide, TGF-beta receptor, and TGF-beta1 mRNA. Modulation of mannose-6-phosphate levels may provide a mean of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

[Effects of platelet-rich plasma on BMSCs differentiation into SC in vitro].

OBJECTIVE: To explore effect of platelet-rich plasma (PRP) on rabbit BMSCs differentiation into SC in vitro and to detect secretory function of the differentiated cells. METHODS: BMSCs isolated from 5 mL bone marrow of 2-month-old New Zealand white rabbit were cultured using density gradient centrifugation and adherence screening methods. A total of 5 mL femoral vein blood was obtained from rabbits to prepare PRP using modified Appel method. The BMSCs at passage 3 were divided into three groups: the combined induction group, in which the cells were cultured with complete medium containing PRP after beta-mercaptoethanol and retinoic acid inductions; the simple induction group, in which the cells were cultured with L-DMEM complete medium without PRP after beta -mercaptoethanol and retinoic acid induction; the control group, in which the cells were cultured with L-DMEM complete medium. Growth condition of the cells in each group was observed using inverted microscope. cell identification was conducted at 4, 7, 9, and 11 days after culture using immunofluorescence staining method, and NGF content was detected by ELISA method. NGF mRNA expression was assayed by RT-PCR 11 days after culture. RESULTS: Most cells in the combined induction and the simple induction group were out of BMSCs typical cell morphology 4 days after culture; cells in the combined induction group were out of BMSCs typical cell morphology and changed into cells resembling SC in terms of morphology and contour 9 days after culture. The cells in the control group showed no obvious morphological changes. S-100 protein expression in the cells was evident in the combined induction and the simple induction group at each time point after induced culture; the positive expression rate of cell in each group was increased over time, and significant differences were evident between the combined induction group and the simple induction group 7, 9, and 11 days after culture (P < 0.05). Control group was negative for the expression. There were significant differences when comparing the control group with the combined induction group or the simple induction group in terms of NGF content at each time point (P < 0.01). Significant difference was evident between the combined induction group and the simple induction group 7, 9, and 11 days after culture (P < 0.05), and no significant difference was noted 4 days after culture (P > 0.05). Relative intensity of NGF mRNA expression in the combined induction group was greater than that of the simple induction group 11 days after culture (P < 0.05). CONCLUSION: Rabbit BMSCs can differentiate into SC excreting NGF under certain induction condition in vitro. PRP can remarkably promote BMSCs differentiation into SC.

[Effects of TGF-beta1 neutralizing antibody on collagen production and adhesion formation of flexor tendon].

OBJECTIVE: To investigate the preventive effect of TGF-beta1 neutralizing antibody on collagen production and adhesion formation of flexor tendon. METHODS: Tendon fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from 6 New Zealand rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-beta along with increasing dose of TGF-beta1 neutralizing antibody. Col I production was measured by enzyme-linked immunoabsorbent assay after 3 days. Eighty-four adult New Zealand White rabbits forepaws underwent sharp transection of middle digit flexor digitorum profundus and immediate repair. Then the rabbits were divided into three groups: the normal saline (NS group, n = 36), 1.0 microg/mL TGF-beta1 neutralizing antibody (1.0 microg/mL TGF-beta1 group, n = 36) and 2.0 microg/mL TGF-beta1 neutralizing antibody (2.0 microg/mL TGF-beta1 group, n = 12) were injected in tendon sheath respectively. Tendons were harvested at 4 and 8 weeks for biomechanics testing, histological evaluation and scanning electron microscope observation. Tendons were harvested at 1, 2, 4 and 8 weeks to determine the mRNA expression of TGF-beta1 and Col I by in situ hybridization. RESULTS: ELISA exhibed that TGF-beta1 enhanced Col I production and the neutralizing antibody significantly inhibited TGF-beta1-induced Col I production in all 3 cell cultures with a dose-dependent. At 4 and 8 weeks after operation the gliding excursion of the tendon and the simulated active flexion in NS group were less than that of 1.0 microg/mL TGF-beta1 group and 2.0 microg/mL TGF-beta1 group. There was significant difference between NS group and 1.0 microg/mL TGF-beta1 group, 2.0 microg/mL TGF-beta1 group (P < 0.05). The tendon anastomosis breaking strength showed no significant differences among three groups (P > 0.05). Scanning electron microscope and histological observation showed that collagen fibers arranged irregularly in NS group, but arranged regularly in 1.0 microg/mL TGF-beta1 group and 2.0 microg/mL TGF-beta1 group at 4 and 8 weeks after operation. The in situ hybridization results revealed that TGF-beta1 and Col I mRNA expression in 1.0 microg/mL TGF-beta1 group was lower than that in NS group at each time. There was significant difference between two groups (P < 0.05). CONCLUSION: TGF-beta1 neutralizing antibody can inhibit the function of the TGF-beta1 effectively and prevent adhesion formation after the flexor tendon injured and repaired.

Anterior cruciate ligament reconstruction with bone-patellar tendon-bone autograft versus allograft.

PURPOSE: The purpose of this study was to analyze the clinical outcome of arthroscopic anterior cruciate ligament (ACL) reconstruction with bone-patellar tendon-bone autograft versus allograft. METHODS: Between May 2000 and June 2004, 172 patients undergoing arthroscopic bone-patellar tendon-bone ACL reconstruction were prospectively randomized into autograft (n = 86) or allograft (n = 86) groups. The senior surgeon performed all operations using the same surgical technique. Each fixation was performed by means of an interference screw. Patients were evaluated preoperatively and postoperatively at follow-up. Of the patients, 156 (76 in the autograft group and 80 in the allograft group) were available for full evaluation. Evaluations included a detailed history, physical examination, functional knee ligament testing, KT-2000 arthrometer testing (MEDmetric, San Diego, CA), Harner's vertical jump and Daniel's 1-leg hop tests, Lysholm score, Tegner score, International Knee Documentation Committee standard evaluation form, Cincinnati knee score, and radiograph. RESULTS: Demographic data were comparable between groups. The mean follow-up was 5.6 years for both groups. There were no statistically significant differences according to evaluations of outcome between the 2 groups except that patients in the allograft group had a shorter operation time and longer fever time postoperatively compared with the autograft group. The postoperative infection rates were 0% and 1.25% for the autograft group and allograft group, respectively. There was a significant difference (P < .05) in the development of osteoarthritis between the operated knee in comparison to the contralateral knee according to radiographs. However, no significant difference was found between the 2 groups at the final follow-up examination (P > .05). CONCLUSIONS: Both groups of patients achieved almost the same satisfactory outcomes after a mean of 5.6 years of follow-up. Allograft is a reasonable alternative to autograft for ACL reconstruction. LEVEL OF EVIDENCE: Level II, prospective comparative study.

ACL reconstruction with BPTB autograft and irradiated fresh frozen allograft.

OBJECTIVE: To analyze the clinical outcomes of arthroscopic anterior cruciate ligament (ACL) reconstruction with irradiated bone-patellar tendon-bone (BPTB) allograft compared with non-irradiated allograft and autograft. METHODS: All BPTB allografts were obtained from a single tissue bank and the irradiated allografts were sterilized with 2.5 mrad of irradiation prior to distribution. A total of 68 patients undergoing arthroscopic ACL reconstruction were prospectively randomized consecutively into one of the two groups (autograft and irradiated allograft groups). The same surgical technique was used in all operations done by the same senior surgeon. Before surgery and at the average of 31 months of follow-up (ranging from 24 to 47 months), patients were evaluated by the same observer according to objective and subjective clinical evaluations. RESULTS: Of these patients, 65 (autograft 33, irradiated allograft 32) were available for full evaluation. When the irradiated allograft group was compared to the autograft group at the 31-month follow-up by the Lachman test, the anterior drawer test (ADT), the pivot shift test, and KT-2000 arthrometer test, statistically significant differences were found. Most importantly, 87.8% of patients in the autograft group and just only 31.3% in the irradiated allograft group had a side-to-side difference of less than 3 mm according to KT-2000. The failure rate of the ACL reconstruction with irradiated allograft (34.4%) was higher than that with autograft (6.1%). The anterior and rotational stabilities decreased significantly in the irradiated allograft group. According to the overall International Knee Documentation Committee (IKDC), functional and subjective evaluations, and activity level testing, no statistically significant differences were found between the two groups. Besides, patients in the irradiated allograft group had a shorter operation time and a longer duration of postoperative fever. When the patients had a fever, the laboratory examinations of all patients were almost normal. Blood routine was normal, the values of erythrocyte sedimentation rate (ESR) were 5~16 mm/h and the contents of C reactive protein (CRP) were 3-10 mg/L. CONCLUSION: We conclude that the short term clinical outcomes of the ACL reconstruction with irradiated BPTB allograft were adversely affected. The less than satisfactory results led the senior authors to discontinue the use of irradiated BPTB allograft in ACL surgery and not to advocate using the gamma irradiation as a secondary sterilizing method.

Anterior cruciate ligament reconstruction with BPTB autograft, irradiated versus non-irradiated allograft: a prospective randomized clinical study.

The effect of using gamma irradiation to sterilize bone-patellar tendon-bone (BPTB) allograft on the clinical outcomes of anterior cruciate ligament (ACL) reconstruction with irradiated allograft remains controversial. Our study was aimed to analyze the clinical outcomes of arthroscopic ACL reconstruction with irradiated BPTB allograft compared with non-irradiated allograft and autograft. All BPTB allografts were obtained from a single tissue bank and the irradiated allografts were sterilized with 2.5 Mrad of irradiation prior to distribution. A total of 102 patients undergoing arthroscopic ACL reconstruction were prospectively randomized consecutively into three groups. The same surgical technique was used in all operations done by the same senior surgeon. Before surgery and at the average of 31 months follow-up (range 24-47 months) patients were evaluated by the same observer according to objective and subjective clinical evaluations. Of these patients, 99 (autograft 33, non-irradiated allograft 34, irradiated allograft 32) were available for full evaluation. When compared the irradiated allograft group to non-irradiated allograft group or autograft group at 31 months follow-up by the Lachman test, ADT, pivot shift test and KT-2000 arthrometer testing, statistically significant differences were found. Most importantly, 87.8% of patients in the Auto group, 85.3% in the Non-Ir-Auto group and just only 31.3% in the Ir-Allo group had a side-to-side difference of less than 3 mm according to KT-2000. The failure rate of the ACL reconstruction with irradiated allograft (34.4%) was higher than that with autograft (6.1%) and non-irradiated allograft (8.8%). The anterior and rotational stability decreased significantly in the irradiated allograft group. According to the overall IKDC, functional, subjective evaluations and activity level testing, no statistically significant differences were found between the three groups. However, there was a trend that the functional and activity level decreased and the patients felt uncomfortable more often in the irradiated allograft group. The statistical analysis showed no significant difference between the non-irradiated allograft group and the autograft group according to the aforementioned evaluations, except that patients in the allograft group had a shorter operation time and a longer duration of postoperative fever. When comparing the postoperative duration of fever of the two allograft groups, there was also a trend that the irradiated allograft group was longer than the non-irradiated allograft group, but no significant difference was found. When the patients had a fever, the laboratory examinations of all patients were almost normal (Blood routine was normal, the values of ESR were 5 - 16 mm/h, CRP were 3 - 10 mg/l). On the basis of our study, we concluded that patients undergoing ACL reconstruction with BPTB non-irradiated allograft or autograft had similar clinical outcomes. Non-irradiated BPTB allograft is a reasonable alternative to autograft for ACL reconstruction. While the short term clinical outcomes of the ACL reconstruction with irradiated BPTB allograft were adversely affected with an increased failure rate. The less than satisfactory results led the senior authors to discontinue the use of irradiated BPTB allograft in ACL surgery and not to advocate that gamma irradiation be used as a secondary sterilizing method. Further research into alternatives to gamma irradiation is needed.

[Gene expression of transforming growth factor beta1 in zone II flexor tendon wound healing of rabbit].

OBJECTIVE: To research the gene expression of transforming growth factor beta1 (TGF-beta1) in zone II flexor tendon wound healing of rabbit. METHODS: Sixty New Zealand white rabbits forepaws (left side) underwent complete transection and the middle digit flexor digitorum profundus tendon in zone II were repaired by Kessler methods as the experimental group. The normal right forepaws served as the control group. The tendons and tendon sheaths were harvested at 1, 7, 14, 21, 28 and 56 days after repair (n = 10). The expression patterns of TGF-beta1 were analyzed by in situ hybridization and immunohistochemistry staining methods. RESULTS: The in situ hybridization examination revealed that TGF-beta1 mRNA expression up-regulated at 1 day, reached the peak levels at 14-21 days and remained high levels up to 56 days in the experimental group. The expression of TGF-beta1 mRNA in control group was lower than that in the experimental group, showing statistically significant difference (P < 0.05). The results of immunohistochemical staining was similar to that of in situ hybridization. CONCLUSION: The normal tendon and tendon sheath cells are capable of TGF-beta1 production. The cytokine is activated in tendon wound condition. The up-regulation of this cytokine in both tendon and tendon sheath cells are coincidence with both extrinsic and intrinsic mechanisms for tendon repair.

99Tcm-DISIDA hepatobiliary imaging in evaluating gallbladder function in patients with spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is associated with increased prevalence of gallstones and acute acalculous cholecystitis. A possible explanation for the increased prevalence of gallstones in SCI patients is decreased gallbladder motility causing gallbladder stasis. In this study, we investigated gallbladder function in patients with SCI. METHODS: Eighteen normal controls, 16 trauma controls and 46 SCI patients were included in this study. Gallbladder function was measured by technium 99m-labeled imino-diacetic acid analogue ((99)Tc(m)-DISIDA) hepatobiliary imaging and represented by filling fraction (FF) and ejection fraction (EF). The data from SCI patients were analyzed according to old versus young, female versus male, heavy versus light body weight, ASIA A & B versus ASIA C & D classification, high- versus low-level injury, and long versus short injury duration. RESULTS: Fifty-two percent of SCI patients had abnormal FF and 59% had abnormal EF. Significantly decreased FF and EF values were found in SCI patients, especially in female patients with severe and high-level injuries. CONCLUSION: Quantitative (99)Tc(m)-DISIDA cholescintigraphy showed that SCI can significantly impair gallbladder function.

Experimental study on low intensity ultrasound and tissue engineering to repair segmental bone defects.

In order to evaluate the efficacy of low intensity ultrasound and tissue engineering technique to repair segmental bone defects, the rabbit models of 1.5-cm long rabbit radial segmental osteoperiosteum defects were established and randomly divided into 2 groups. All defects were implanted with the composite of calcium phosphate cement and bone mesenchymal stem cells, and additionally those in experimental group were subjected to low intensity ultrasound exposure, while those in control group to sham exposure. The animals were killed on the postoperative week 4, 8 and 12 respectively, and specimens were harvested. By using radiography and the methods of biomechanics, histomorphology and bone density detection, new bone formation and material degradation were observed. The results showed that with the prolongation of time after operation, serum alkaline phosphatase (AKP) levels in both groups were gradually increased, especially in experimental group, reached the peak at 6th week (experimental group: 1.26 mmol/L; control group: 0.58 mmol/L), suggesting the new bone formation in both two group, but the amount of new bone formation was greater and bone repairing capacity stronger in experimental group than in control group. On the 4th week in experimental group, chondrocytes differentiated into woven bone, and on the 12th week, remodeling of new lamellar bone and absorption of the composite material were observed. The mechanical strength of composite material and new born density in experimental group were significantly higher than in control group, indicating that low intensity ultrasound could not only effectively increase the formation of new bone, but also accelerate the calcification of new bone. It was concluded that low intensity ultrasound could evidently accelerate the healing of bone defects repaired by bone tissue engineering.