Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model.

Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injuries in which tissue loss results in permanent functional impairment, is common among these injuries. The present standard of care entails the use of muscle flap transfers, which suffer from the need for additional surgery when using autografts or the risk of rejection when cadaveric grafts are used. Tissue engineering (TE) strategies for skeletal muscle repair have been investigated as a means to overcome current therapeutic limitations. In that regard, human hair-derived keratin (KN) biomaterials have been found to possess several favorable properties for use in TE applications and, as such, are a viable candidate for use in skeletal muscle repair. Herein, KN hydrogels with and without the addition of skeletal muscle progenitor cells (MPCs) and/or insulin-like growth factor 1 (IGF-1) and/or basic fibroblast growth factor (bFGF) were implanted in an established murine model of surgically induced VML injury to the latissimus dorsi (LD) muscle. Control treatments included surgery with no repair (NR) as well as implantation of bladder acellular matrix (BAM). In vitro muscle contraction force was evaluated at two months postsurgery through electrical stimulation of the explanted LD in an organ bath. Functional data indicated that implantation of KN+bFGF+IGF-1 (n = 8) enabled a greater recovery of contractile force than KN+bFGF (n = 8)***, KN+MPC (n = 8)**, KN+MPC+bFGF+IGF-1 (n = 8)**, BAM (n = 8)*, KN+IGF-1 (n = 8)*, KN+MPCs+bFGF (n = 9)*, or NR (n = 9)**, (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent with the physiological findings, histological evaluation of retrieved tissue revealed much more extensive new muscle tissue formation in groups with greater functional recovery (e.g., KN+IGF-1+bFGF) when compared with observations in tissue from groups with lower functional recovery (i.e., BAM and NR). Taken together, these findings further indicate the general utility of KN biomaterials in TE and, moreover, specifically highlight their potential application in the treatment of VML injuries.

Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries exceed the considerable intrinsic regenerative capacity of skeletal muscle, resulting in permanent functional and cosmetic deficits. VML and VML-like injuries occur in military and civilian populations, due to trauma and surgery as well as due to a host of congenital and acquired diseases/syndromes. Current therapeutic options are limited, and new approaches are needed for a more complete functional regeneration of muscle. A potential solution is human hair-derived keratin (KN) biomaterials that may have significant potential for regenerative therapy. The goal of these studies was to evaluate the utility of keratin hydrogel formulations as a cell and/or growth factor delivery vehicle for functional muscle regeneration in a surgically created VML injury in the rat tibialis anterior (TA) muscle. VML injuries were treated with KN hydrogels in the absence and presence of skeletal muscle progenitor cells (MPCs), and/or insulin-like growth factor 1 (IGF-1), and/or basic fibroblast growth factor (bFGF). Controls included VML injuries with no repair (NR), and implantation of bladder acellular matrix (BAM, without cells). Initial studies conducted 8 weeks post-VML injury indicated that application of keratin hydrogels with growth factors (KN, KN+IGF-1, KN+bFGF, and KN+IGF-1+bFGF, n = 8 each) enabled a significantly greater functional recovery than NR (n = 7), BAM (n = 8), or the addition of MPCs to the keratin hydrogel (KN+MPC, KN+MPC+IGF-1, KN+MPC+bFGF, and KN+MPC+IGF-1+bFGF, n = 8 each) (p < 0.05). A second series of studies examined functional recovery for as many as 12 weeks post-VML injury after application of keratin hydrogels in the absence of cells. A significant time-dependent increase in functional recovery of the KN, KN+bFGF, and KN+IGF+bFGF groups was observed, relative to NR and BAM implantation, achieving as much as 90% of the maximum possible functional recovery. Histological findings from harvested tissue at 12 weeks post-VML injury documented significant increases in neo-muscle tissue formation in all keratin treatment groups as well as diminished fibrosis, in comparison to both BAM and NR. In conclusion, keratin hydrogel implantation promoted statistically significant and physiologically relevant improvements in functional outcomes post-VML injury to the rodent TA muscle.

Further development of a tissue engineered muscle repair construct in vitro for enhanced functional recovery following implantation in vivo in a murine model of volumetric muscle loss injury.

Volumetric muscle loss (VML) can result from trauma and surgery in civilian and military populations, resulting in irrecoverable functional and cosmetic deficits that cannot be effectively treated with current therapies. Previous work evaluated a bioreactor-based tissue engineering approach in which muscle derived cells (MDCs) were seeded onto bladder acellular matrices (BAM) and mechanically preconditioned. This first generation tissue engineered muscle repair (TEMR) construct exhibited a largely differentiated cellular morphology consisting primarily of myotubes, and moreover, significantly improved functional recovery within 2 months of implantation in a murine latissimus dorsi (LD) muscle with a surgically created VML injury. The present report extends these initial observations to further document the importance of the cellular phenotype and composition of the TEMR construct in vitro to the functional recovery observed following implantation in vivo. To this end, three distinct TEMR constructs were created by seeding MDCs onto BAM as follows: (1) a short-term cellular proliferation of MDCs to generate primarily myoblasts without bioreactor preconditioning (TEMR-1SP), (2) a prolonged cellular differentiation and maturation period that included bioreactor preconditioning (TEMR-1SPD; identical to the first generation TEMR construct), and (3) similar treatment as TEMR-1SPD but with a second application of MDCs during bioreactor preconditioning (TEMR-2SPD); simulating aspects of "exercise" in vitro. Assessment of maximal tetanic force generation on retrieved LD muscles in vitro revealed that TEMR-1SP and TEMR-1SPD constructs promoted either an accelerated (i.e., 1 month) or a prolonged (i.e., 2 month postinjury) functional recovery, respectively, of similar magnitude. Meanwhile, TEMR-2SPD constructs promoted both an accelerated and prolonged functional recovery, resulting in twice the magnitude of functional recovery of either TEMR-1SP or TEMR-1SPD constructs. Histological and molecular analyses indicated that TEMR constructs mediated functional recovery via regeneration of functional muscle fibers either at the interface of the construct and the native tissue or within the BAM scaffolding independent of the native tissue. Taken together these findings are encouraging for the further development and clinical application of TEMR constructs as a VML injury treatment.

Exercise improves bladder function in diabetic mice.

AIMS: We determined the effect of exercise on bladder dysfunction and voiding frequency in db/db mice. MATERIALS AND METHODS: Diabetic db/db female mice (BKS.Cg-Dock7m +/+ Leprdb/J strain) and their age-matched wild-type controls (WT) were equally divided into sedentary and exercise groups. Mice were exercised for 1 hr everyday for 8 weeks (speed of 5.2 m/min). We performed a voiding pattern test, cystometric analysis and reactivity of isolated bladder strips in WT and db/db mice, both sedentary and exercised. RESULTS: Diabetes increased the frequency of voiding, bladder capacity, and residual volume. Exercise decreased voiding frequency in db/db mice; voiding frequency was 5.8 ± 0.5 (db/db exercise) versus 10.8 ± 1.1 (db/db control, P < 0.001). In cystometric analysis, the bladder capacity of db/db sedentary mice was 0.27 ± 0.05 ml and was 0.14 ± 0.02 ml in the db/db exercise group (P < 0.05), whereas the residual volume was 0.2 ± 0.03 ml in db/db sedentary mice and 0.06 ± 0.02 ml in db/db Ex mice. Isolated strips of bladder muscle from sedentary db/db mice were more responsive to carbachol than strips from db/db exercise mice. Exercise did not improve the urodynamic properties of WT mice, both sedentary and exercised. CONCLUSIONS: Exercise improves bladder function in diabetic mice by reducing voiding frequency and improving urodynamic parameters.