Leung Kim Hung

Increased apoptosis at the late stage of tendon healing

The mechanism for the clearance of excess healing fibroblasts at the end of tendon healing has not been reported despite the importance of maintaining tissue homeostasis. This study investigated the role of apoptosis in cell turnover in a rat central 1/3 patellar tendon donor site injury model. At days 4, 7, 14, 28, months 2 and 6, the rats were killed. Patellar tendons without injury served as control. Apoptotic cells were determined by an in situ terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labeling (TUNEL) assay and anti‐active caspase‐3 antibodies, while proliferating cells were determined by anti‐proliferating cell nuclear antigen antibodies. The total fibroblast‐like cell density in the center of the wound increased from day 4 and thereafter steadily returned to normal. In situ TUNEL assay showed few positive staining cells in the wound at days 4 and 7. The percentages of TUNEL‐positive fibroblast‐like cells showing morphological characteristics of apoptosis increased sharply and reached the maximum on day 28 (median %: 31.38%). No fibroblast‐like cell was stained at month 6 and the healed tissue was similar to that in a normal uninjured tendon. A similar trend was observed with active caspase‐3 immunohistochemistry. In conclusion, an increase in apoptosis at the end of tendon healing coincided with a decrease in cellularity.

Effect of anti-osteoporotic agents on the prevention of bone loss in unloaded bone

Pharmaceutical countermeasures to treat disuse osteoporosis are rarely studied. Pharmaceutical studies for the treatment and prevention of osteoporosis depend on the ovariectomized rat model, which is a suitable model for the disease in women. Disuse osteoporosis affects men and women, but there is lack of awareness and relevant pharmaceutical studies for this condition. The objectives of this study were to verify the validity of an unusual tail-suspension rat model in the induction of disuse osteoporosis and subsequent pharmaceutical treatments. This model was created by unloading the hind limbs of the rats in order to create a state of weightlessness in their hindlimb bones. Validation of the model was performed with non-suspended rats. This study included five groups of suspended rats fed with different agents, such as distilled water (control), high-, medium- and low-dose raloxifene and a bisphosphonate (alendronate). The experiment lasted for 28 days. Comparisons were made between the suspended control and treatment groups. Ovariectomized and sham‑operated rats were also included as a reference for bone changes during osteoporosis. Changes in bone mineral density (BMD) at the distal femur and proximal tibia, microarchitecture at the distal femur and biomechanical strength at the diaphyseal femur were studied. Reduction of BMD and deterioration of trabeculae were similar between the suspended control and ovariectomized rats. Loss of BMD induced by tail suspension was reduced most effectively by medium-dose raloxifene. Deterioration of trabecular microarchitecture was also prevented by raloxifene. The tail-suspension rat model is suitable for the study of disuse osteoporosis under the effects of various therapeutic agents. The preventive effects of raloxifene against bone loss under disuse conditions have been demonstrated using this model.

Rehabilitation Using Topical Agent—Myth or Reasonable Option?

Objective To investigate whether topical agents of herbal origin used in traditional Chinese medicine (TCM) have real biological effects on limb swelling and tissue healing. Methods Formation of an innovative formula was achieved through a literature screening of over 200 herbs. The herbs selected have been reported to be anti-inflammatory, promoting circulation and supporting tissue/bone healing. In vitro and in vivo tests were done to investigate the biological effects of the herbs. Results Topical agent for tissue swelling: (a) The herbs were found to facilitate the transport of a chemical through an artificial membrane and porcine skin in a diffusion chamber; (b) the herbal bath was found to positively reduce hand and foot swelling. Topical agent for fracture healing: (a) In vitro tests showed positive anti-inflammatory, circulation promotion and tissue healing effects of a six herbs formula; (b) treated animals showed bigger callus, higher serum bone specific alkaline phosphatase and better mechanical strength; (c) liquid chromatography-mass spectrometry testing showed that there was a real transport of chemical markers across the skin. Conclusion TCM herbal agents used topically for hand swelling and tissue healing do have demonstrable scientific basis. Further research is justified.

Do We Need to Repair the Nerves When Replanting Distal Finger Amputations

Distal replantation is an excellent model to study the results of nerve repair. We aim to demonstrate differences in aesthetic, sensory, and functional outcomes in fingertip replantation, with and without nerve repair. We recruited 28 fingers in 28 patients, who had successful distal replantation in 5 years. Half of the fingers had nerves repaired. Mean follow-up was 39 months. Symptoms of pain, numbness, cold intolerance, scar hypersensitivity, pulp atrophy, and weakness were reported. Nail width, pulp length, 2-point discrimination, Semmes-Weinstein test, and power were evaluated. We used chi-square tests of independence to examine association between nerve repair and symptoms, and independent T tests and Mann-Whitney U tests to analyze difference between replantation with and without nerve repair according to objective results. Chi-square tests reviewed no significant association between nerve repair and symptoms. Mann-Whitney U tests showed no significant difference between the groups, with and without nerve repair. All fingers showed mean 2-point discrimination of 5.6 mm, and Semmes-Weinstein test results of green in 3 fingers and blue in 17. There was no significant difference in overall outcomes in repairing nerve or not in distal finger replantation. Both groups had satisfactory outcomes. Possibly, spontaneous neurotization is present, and nerve repair is not necessary, which may help to shorten the operation time and decrease extensiveness of surgeries.

Effects of Redox Modulation on Cell Proliferation, Viability, and Migration in Cultured Rat and Human Tendon Progenitor Cells

Tendon healing is slow and usually results in inferior fibrotic tissue formation. Recently, application of tendon derived stem cells (TDSCs) improved tendon healing in animal studies. In a chicken model, local injection of antioxidants reduced tendon adhesion after tendon injury. An in vitro study demonstrated that supplementation of H2O2 reduced tenogenic marker expression in TDSCs. These findings suggested that the possibility of TDSCs is involved in tendon healing and the cellular activities of TDSCs might be affected by oxidative stress of the local environment. After tendon injury, oxidative stress is increased. Redox modulation might affect healing outcomes via affecting cellular activities in TDSCs. To study the effect of oxidative stress on TDSCs, the cellular activities of rat/human TDSCs were measured under different dosages of vitamin C or H2O2 in this study. Lower dose of vitamin C increased cell proliferation, viability and migration; H2O2 affected colony formation and suppressed cell migration, cell viability, apoptosis, and proliferation. Consistent with previous studies, oxidative stresses (H2O2) affect both recruitment and survival of TDSCs, while the antioxidant vitamin C may exert beneficial effects at low doses. In conclusion, redox modulation affected cellular activities of TDSCs and might be a potential strategy for tendon healing treatment.

Local administration of Trolox, a vitamin E analog, reduced tendon adhesion in a chicken model of flexor digitorum profundus tendon injury

Summary Background Hand flexor tendon injuries are compromised with tendon adhesion. Tendon adhesion forms between flexor tendon and tendon sheath, reduces the range of motion of fingers, and affects their function. Oxidative stress is increased in flexor tendon after injury and might play a role in tendon adhesion formation. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analog of vitamin E, is antioxidative. Trolox reduced oxidative stress and the expression of fibrotic cytokines in the bile gut ligation animal model. Vitamin C and Trolox are strong antioxidants, but they might also have prooxidant properties. The prooxidant properties of vitamin C and Trolox are different. In this study, our aim was to determine the effect of Trolox in reducing tendon adhesion formation. Methods Flexor digitorum profundus tendon injury was induced in 54 Kai-Mei Chicken according to a well-established protocol. After wound closure, an injection of 50 μL saline, 10mM Trolox, or 100mM Trolox was administered into the wound area. At 2 weeks or 6 weeks after the surgery, chicken feet were harvested for gliding test, high-resolution ultrasound measurement on a fibrotic area, and histology. Results At Week 2 after the surgery, Trolox has no effect on the flexion angle and gliding resistance, whereas a significant improvement was observed in the flexion angle and gliding resistance in the Trolox-treated groups at Week 6. However, no dose response was observed. In the ultrasound measurement, there was no significant difference in the fibrotic mass in the Trolox-treated group as compared to the saline group at Week 2. At Week 6, fibrotic mass was significantly reduced in both Trolox-treated groups. From the histological examination, the Trolox-treated groups presented a higher cellularity at Week 2 as compared to the saline group, and reduced fibrosis and adhesion at Week 6. Conclusion Our results suggest that local administration of Trolox can reduce tendon adhesion, and a higher dose of Trolox did not have negative effects. Clinical Significance Trolox solution might be feasible to reduce tendon adhesion via intraoperative injection at the wound area during tendon repair.

Evaluation of a Topical Herbal Agent for the Promotion of Bone Healing

A topically used Chinese herbal paste, namely, CDNR, was designed to facilitate fracture healing which is usually not addressed in general hospital care. From our in vitro studies, CDNR significantly inhibited the release of nitric oxide from RAW264.7 cells by 51 to 77%. This indicated its anti-inflammatory effect. CDNR also promoted the growth of bone cells by stimulating the proliferation of UMR106 cells up to 18%. It also increased the biomechanical strength of the healing bone in a drill-hole defect rat model by 16.5% significantly. This result revealed its in vivo efficacy on facilitation of bone healing. Furthermore, the detection of the chemical markers of CDNR in the skin and muscle of the treatment area demonstrated its transdermal properties. However, CDNR did not affect the bone turnover markers in serum of the rats. With its anti-inflammatory and bone formation properties, CDNR is found effective in promoting bone healing.

In vitro Biomechanical Study of Pulvertaft Tendon Weaving Technique

Background/Purposes: The outcome of tendon repair depends on the strength, which allows early active mobilization to achieve better function without rupture. The aims of this study are to assess quantitatively the biomechanical properties and relationship between the number of tendon weaving and suture method using Pulvertaft technique. Results: We found that the load to failure was increased with increasing number of weaves and sutures. From 1-weave to 4-weave single suture samples, the peak load to failure was 9.5 N, 19.7 N, 37.5 N, and 42.6 N, respectively. Based on previous studies, wrist and finger tendons should withstand 1e8 N on passive mobilization. Conclusion: On active mobilization, finger tendon repair need to provide 34 N for immediate mobilization. Therefore, irrespective of number of sutures, both 3and 4-weave repairs could allow early mobilization biomechanically.