Flexor tendon wound healing in vitro: lactate up-regulation of TGF-beta expression and functional activity

Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.     

Differential expression of transforming growth factor-beta receptors in a rabbit zone II flexor tendon wound healing model

Flexor tendon repair in zone II is complicated by adhesions that impair normal postoperative gliding. Transforming growth factor-beta (TGF-beta) is a family of growth factors that has been implicated in scar formation. The TGF-beta family of proteins binds to three distinct classes of membrane receptors, termed RI, RII, and RIII. In this study, we analyzed the temporal and spatial distribution of TGF-beta receptor isoforms (RI, RII, and RIII) in a rabbit zone II flexor tendon wound healing model.Twenty-eight adult New Zealand White rabbit forepaws underwent isolation of the middle digit flexor digitorum profundus tendon in zone II. The tendons underwent transection in zone II and immediate repair. The tendons were harvested at increasing time points: 1, 3, 7, 14, 28, and 56 days postoperatively (n = 4 at each time point). The control flexor tendons were harvested without transection and repair (n = 4). Immunohistochemical analysis was used to detect the expression patterns for TGF-beta receptors RI, RII, and RIII.Immunohistochemical staining of the transected and repaired tendons demonstrated up-regulation of TGF-beta RI, RII, and RIII protein levels. TGF-beta receptor production in the experimental group (transection and repair) was concentrated in the epitenon and along the repair site. Furthermore, the TGF-beta receptor expression levels peaked at day 14 and decreased by day 56 postoperatively. In contrast, minimal receptor expression was observed in the untransected and unrepaired control tendons.These data provide evidence that (1) TGF-beta receptors are up-regulated after injury and repair; (2) peak levels of TGF-beta receptor expression occurred at day 14 and decreased by day 56 after wounding and repair; and (3) both the tendon sheath and epitenon have the highest receptor expression, and both may play critical roles in flexor tendon wound healing. Understanding the up-regulation of TGF-beta isoforms and the up-regulation of their corresponding receptors during flexor tendon wound healing provides new targets for biomolecular modulation of postoperative scar formation.     

Gender specific effects on the actin-remodelling protein Flightless I and TGF-beta1 contribute to impaired wound healing in aged skin

Impaired wound healing in the elderly presents a major clinical challenge. Understanding the cellular mechanisms behind age-related impaired healing is vital for developing new wound therapies. Here we show that the actin-remodelling protein, Flightless I (FliI) is a contributing factor to the poor healing observed in elderly skin and that gender plays a major role in this process. Using young and aged, wild-type and FliI overexpressing mice we found that aging significantly elevated FliI expression in the epidermis and wound matrix. Aging exacerbated the negative effect of FliI on wound repair and wounds in aged FliI transgenic mice were larger with delayed reepithelialisation. When the effect of gender was further analysed, despite increased FliI expression in young and aged male and female mice, female FliI transgenic mice had the most severe wound healing phenotype suggesting that male mice were refractory to FliI gene expression. Of potential importance, males, but not females, up-regulated transforming growth factor-β1 and this was most pronounced in aged male FliI overexpressing wounds. As FliI also functions as a co-activator of the estrogen nuclear receptor, increasing concentrations of β-estradiol were added to skin fibroblasts and keratinocytes and significantly enhanced FliI expression and translocation of FliI from the cytoplasm to the nucleus was observed. FliI further inhibited estrogen-mediated collagen I secretion suggesting a mechanism via which FliI may directly affect provisional matrix synthesis. In summary, FliI is a contributing factor to impaired healing and strategies aimed at decreasing FliI levels in elderly skin may improve wound repair.     

A fine structural study of the development of the chick flexor digital tendon: a model for synovial sheathed tendon healing.

The development of the synovial sheathed flexor digital tendon in the chick was studied by light and electron microscopy in 12-day embryos to 22-day post-hatched chickens. Areas of specialized connective tissue differentiation were identified in this complex structure consisting of a lubricated synovial sheath, elastic vincula and fibrocartilaginous adaptations on the surface of the tendon. The presence of some of these specialized adaptations may be related to the specific types of mechanical forces and stresses applied to the developing connective tissue system. This model system appears to be appropriate for the experimental study of tendon injuries related to the human hand.     

Adding weights to stretching exercise increases passive range of motion for healthy elderly

Stretching exercise is effective for increasing joint range of motion (ROM). However, the Surgeon General's Report and the American College of Sports Medicine cite a lack of studies identifying strategies capable of increasing the effectiveness of stretching exercise. This investigation evaluated adding modest weight (0.45-1.35 kg) to a stretching exercise routine (Body Recall [BR]) on joint ROM. Forty-three subjects ages 55-83 years participated in 1 of 2 training groups, BR, BR with weights (BR+W), or a control group (C). ROM was evaluated at the neck, shoulder, hip, knee, and ankle before and after 10 weeks of exercise. Using ANCOVA, significant differences (p < 0.01) were observed for right and left cervical rotation, hip extension, ankle dorsiflexion, ankle plantar flexion, and shoulder flexion. Post hoc analysis revealed that cervical rotation (left and right), hip extension, and ankle dorsiflexion for BR+W subjects differed significantly from BR and C (p < 0.01). Significant differences with shoulder flexion and ankle plantar flexion were found for both BR and BR+W in comparison to C (p < 0.01). Results indicate that addition of weights enhanced the effectiveness of stretching exercise for increasing joint ROM with 4 of the 6 selected measurements. Thus, a modest intensity exercise program that is within the reach of most elderly may significantly affect joint ROM and flexibility.     

Broadly neutralizing anti-HBV antibody binds to non-epitope regions of preS1

Broadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.     

Whole body vibration increases area and stiffness of the flexor carpi ulnaris tendon in the rat

Whole body vibration (WBV) has been extensively studied as an anabolic stimulus for bone and muscle. Therapeutic WBV delivers low magnitude, high frequency vibrations to tissues, eliciting biological and structural responses. This study investigated the effect of 0.3G (Peak-to-Peak), 30Hz sinusoidal vibration on intact flexor carpi ulnaris tendons in rats. Experimental rats were subjected to twenty minutes of WBV daily for five days a week for a total of five weeks. The tendon cross-sectional area and the structural properties of the muscle-tendon-bone unit under tensile loading to failure were evaluated. Initial body weights were similar between the groups and the mean change in body weight of the animals of each group did not differ. The cross-sectional area of the tendons of the vibrated animals was found to be 32% greater (P<0.05) than the controls and the structural stiffness of the vibrated tendons was found to be 41% greater (P<0.05) than the controls. For specimens that failed in the midsubstance of the tendon, a trend (P=0.087) for increased ultimate load was observed in the vibrated tendons compared to the controls. No differences in material properties were observed except for the strain to ultimate load, which was reduced 22% in the vibrated group. These initial findings suggest that vibration may serve as an anabolic stimulus to tendon similar to its effects on bone and muscle. These findings are important as they open the potential that low magnitude, high frequency vibration might serve as a means to accelerate tendon healing.     

A combined regimen of controlled motion following flexor tendon repair in "no man's land"

A program of controlled motion following repair of flexor tendons in the hand is presented. This regimen incorporates the features of active extension against rubber band passive flexion, as well as those of controlled passive extension and passive flexion. In this prospective study, 44 digits with complete lacerations of the flexor digitorum profundus and flexor digitorum superficialis in zone 2 were treated. Using the Strickland formula of total active motion of the interphalangeal joints, 36 fingers (82 percent) were rated "excellent"; 7 fingers (16 percent) were rated "good"; 1 finger (2 percent) was rated "fair"; none was rated "poor". There was no statistical difference between the results of delayed primary repair and immediate primary repair.     

Efficacy of the designer antimicrobial peptide SHAP1 in wound healing and wound infection

Infected wounds cause delay in wound closure and impose significantly negative effects on patient care and recovery. Antimicrobial peptides (AMPs) with antimicrobial and wound closure activities, along with little opportunity for the development of resistance, represent one of the promising agents for new therapeutic approaches in the infected wound treatment. However, therapeutic applications of these AMPs are limited by their toxicity and low stability in vivo. Previously, we reported that the 19-amino-acid designer peptide SHAP1 possessed salt-resistant antimicrobial activities. Here, we analyzed the wound closure activities of SHAP1 both in vitro and in vivo. SHAP1 did not affect the viability of human erythrocytes and keratinocytes up to 200 μM, and was not digested by exposure to proteases in the wound fluid, such as human neutrophil elastase and Staphylococcus aureus V8 proteinase for up to 12 h. SHAP1 elicited stronger wound closure activity than human cathelicidin AMP LL-37 in vitro by inducing HaCaT cell migration, which was shown to progress via transactivation of the epidermal growth factor receptor. In vivo analysis revealed that SHAP1 treatment accelerated closure and healing of full-thickness excisional wounds in mice. Moreover, SHAP1 effectively countered S. aureus infection and enhanced wound healing in S. aureus-infected murine wounds. Overall, these results suggest that SHAP1 might be developed as a novel topical agent for the infected wound treatment.     

Healthy older adults have insufficient hip range of motion and plantar flexor strength to walk like healthy young adults

Limited plantar flexor strength and hip extension range of motion (ROM) in older adults are believed to underlie common age-related differences in gait. However, no studies of age-related differences in gait have quantified the percentage of strength and ROM used during gait. We examined peak hip angles, hip torques and plantar flexor torques, and corresponding estimates of functional capacity utilized (FCU), which we define as the percentage of available strength or joint ROM used, in 10 young and 10 older healthy adults walking under self-selected and controlled (slow and fast) conditions. Older adults walked with about 30% smaller hip extension angle, 28% larger hip flexion angle, 34% more hip extensor torque in the slow condition, and 12% less plantar flexor torque in the fast condition than young adults. Older adults had higher FCU than young adults for hip flexion angle (47% vs. 34%) and hip extensor torque (48% vs. 27%). FCUs for plantar flexor torque (both age groups) and hip extension angle (older adults in all conditions; young adults in self-selected gait) were not significantly <100%, and were higher than for other measures examined. Older adults lacked sufficient hip extension ROM to walk with a hip extension angle as large as that of young adults. Similarly, in the fast gait condition older adults lacked the strength to match the plantar flexor torque produced by young adults. This supports the hypothesis that hip extension ROM and plantar flexor strength are limiting factors in gait and contribute to age-related differences in gait.     

The VD1 neutralizing antibody to vascular endothelial growth factor-D: binding epitope and relationship to receptor binding

Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that promotes tumor growth and metastatic spread in animal models of cancer. Expression of VEGF-D in prevalent human cancers was reported to correlate with lymph node metastasis and patient outcome—hence, this protein is a potential target for novel anticancer therapeutics designed to restrict tumor growth and spread. Here, we define the binding site in VEGF-D of a neutralizing antibody, designated VD1, which blocks the interaction of VEGF-D with its cell surface receptors vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 and is being used for the development of therapeutic antibodies. We show by peptide-based mapping and site-directed mutagenesis that the VD1 binding site includes the five residues ₁₄₇NEESL₁₅₁ and that immunization with a synthetic peptide containing this motif generates antibodies that neutralize VEGF-D. The tertiary structure of VEGF-D indicates that the ₁₄₇NEESL₁₅₁ epitope is located in the L2 loop of the growth factor, which is important for receptor binding. Mutation of any of these five residues influences receptor binding; for example, mutations to E148, which abolished binding to VD1, impaired the interaction with VEGFR-2 but enhanced binding to VEGFR-3. This structure/function study indicates that the VD1 binding epitope is part of the receptor binding site of VEGF-D, identifies a region of VEGF-D critical for binding of receptors and explains why VD1 does not bind other members of the VEGF family of growth factors.