Comparative host response of 2 human acellular dermal matrices in a primate implant model.

OBJECTIVE: We examined the differences in capsule formation between 2 commercially available human acellular dermal matrices in a nonhuman primate model. METHODS: Primates were implanted dorsally with a subcutaneously placed tissue expander and randomized into 3 groups, receiving skin coverage only, coverage with non-irradiated freeze-dried human acellular dermal matrix, or coverage with gamma-irradiated human acellular dermal matrix. After 9 weeks, soft tissue around the tissue expander was excised and evaluated qualitatively and quantitatively to assess extent of inflammation (CD68 antibodies and interleukin-6 levels), degradation and fibrosis (matrix metalloproteinase-1 and procollagen-1 staining), and mechanical (tensile) strength. RESULTS: Histological evaluation of tissue around the tissue expander indicated differences in host response, suggesting capsule presence in the gamma-irradiated matrix group but not the freeze-dried matrix group. The extent of local inflammation was much higher in the gamma-irradiated matrix group which demonstrated mean (standard deviation) localized interleukin-6 concentration of 67.3 (53.6) vs 16.3 (6.7) pg/mg protein in the non-irradiated matrix group. There was robust degradation and fibrotic response in the gamma-irradiated matrix group versus the freeze-dried matrix group. Mechanical testing indicated mean (standard deviation) ultimate tensile strength of 12.0 (7.1) N in the gamma-irradiated matrix group versus 99.3 (48.8) N in the freeze-dried matrix group. CONCLUSIONS: Enclosure of a tissue expander with human acellular dermal matrix untreated by gamma irradiation led to minimal inflammation and minimal evidence of fibrosis/capsule around the tissue expander compared with robust capsule formation around the tissue expander that was covered by a gamma-irradiated human acellular dermal matrix.

Outcomes after a single-stage procedure for cell-based cartilage repair: a prospective clinical safety trial with 2-year follow-up.

BACKGROUND: There are currently several approaches being pursued to treat focal defects of articular cartilage, each having specific advantages or challenges. A single-stage procedure that uses autologous cartilage fragments, Cartilage Autograft Implantation System (CAIS), is being evaluated in patients and may offer a clinically effective option. PURPOSE: To establish the safety of CAIS and to test whether CAIS improves quality of life by using standardized outcomes assessment tools. STUDY DESIGN: Randomized controlled trial; Level of evidence, 2. METHODS: Patients (n = 29) were randomized (1:2) with the intent to treat with either a control (microfracture [MFX]) or an experimental (CAIS) procedure. Patients were followed at predetermined time points for 2 years using several standardized outcomes assessment tools (SF-36, International Knee Documentation Committee [IKDC], Knee injury and Osteoarthritis Outcome Score [KOOS]). Magnetic resonance imaging was performed at baseline, 3 weeks, and 6, 12, and 24 months. RESULTS: Lesion size and International Cartilage Repair Society (ICRS) grade were similar in both groups. General outcome measures (eg, physical component score of the SF-36) indicated an overall improvement in both groups, and no differences in the number of adverse effects were noted in comparisons between the CAIS and MFX groups. The IKDC score of the CAIS group was significantly higher (73.9 ± 14.72 at 12 months and 82.95 ± 14.88 at 24 months) compared with the MFX group (57.78 ± 18.31 at 12 months and 59.5 ± 13.44 at 24 months). Select subdomains (4/5) in the KOOS instrument were significantly different at 12 and 18 months, and all subdomains (Symptoms and Stiffness, Pain, Activities of Daily Living, Sports and Recreation, Knee-related Quality of Life) were significantly increased at 24 months in CAIS with scores of 88.47 ± 11.68, 90.64 ± 7.87, 97.29 ± 3.8, 78.16 ± 22.06, and 69 ± 23.15 compared with 75 ± 9.31, 78.94 ± 13.73, 89.46 ± 8.13, 51.67 ± 26.01, and 37.15 ± 21.67 in the MFX group. These significant improvements were maintained at 24 months in both IKDC and KOOS. Qualitative analysis of the imaging data did not note differences between the 2 groups in fill of the graft bed, tissue integration, or presence of subchondral cysts. Patients treated with MFX had a significantly higher incidence of intralesional osteophyte formation (54% and 70% of total number of lesions treated) at 6 and 12 months when compared with CAIS (8% and 25% of total number of lesions treated). CONCLUSION: The first clinical experience in using CAIS for treating patients with focal chondral defects indicates that it is a safe, feasible, and effective method that may improve long-term clinical outcomes.

Regeneration of a spinal ligament after total lumbar disk arthroplasty in primates.

Total disk arthroplasty (TDA) is a new procedure that replaces the intervertebral disk space with an artificial motion segment and necessitates the resection of the anterior longitudinal ligament (ALL). We assessed whether a collagen-based graft made from porcine small-intestine submucosa (SIS) can be used as a regenerative scaffold to restore the function and structure of the ALL in the lumbar spine. A total of 10 mature male baboons underwent TDA at L5-L6 using one of two treatments: (1) TDA only (n = 5) or (2) TDA combined with SIS (n = 5). Six months postoperatively, mock revision surgery was performed to assess tissue adhesions followed by non-destructive multidirectional flexibility testing of the spinal segment. The vertebral segments were then processed for histology. The tissue adhesion score was 2.8 +/- 0.8 in the TDA only group and 1.8 +/- 1.4 in the TDA-SIS group (p = 0.2). Segmental range of motion and the length of the neutral zone were similar in both groups. Histology showed that the SIS scaffold led to an organized ligamentous structure with a significantly (p = 0.027) higher thickness (2.18 +/- 0.25 mm) compared to the connective tissue structure in the TDA-only group (1.66 +/- 0.33 mm). We concluded that using a SIS bioscaffold after TDA did not lead to increased great vessel adhesion while its use facilitated the formation of highly organized ligamentous tissues. However, the SIS- induced and newly formed ligamentous tissue anterior to the spinal segment did not lead to a measurable limitation of spinal extension.

In vitro and in vivo comparison of five biomaterials used for orthopedic soft tissue augmentation.

OBJECTIVE: To compare biomaterials used in orthopedics with respect to in vitro cell viability and cell retention and to in vivo tissue healing and regeneration. ANIMALS: 65 adult female Sprague-Dawley rats and synovium, tendon, meniscus, and bone marrow specimens obtained from 4 adult canine cadavers. PROCEDURES: Synovium, tendon, meniscus, and bone marrow specimens were used to obtain synovial fibroblasts, tendon fibroblasts, meniscal fibrochondrocytes, and bone marrow-derived connective tissue progenitor cells for culture on 5 biomaterials as follows: cross-linked porcine small intestine (CLPSI), non-cross-linked human dermis, cross-linked porcine dermis, non-cross-linked porcine small intestine (NCLPSI), and non-cross-linked fetal bovine dermis. After 1 week of culture, samples were evaluated for cell viability, cell density, and extracellular matrix production. Biomaterials were evaluated in a 1-cm(2) abdominal wall defect in rats. Each biomaterial was subjectively evaluated for handling, suturing, defect fit, and ease of creating the implant at the time of surgery, then grossly and histologically 6 and 12 weeks after surgery. RESULTS: All biomaterials allowed for retention of viable cells in culture; however, CLPSI and NCLPSI were consistently superior in terms of cell viability and cell retention. Cell infiltration for NCLPSI was superior to other biomaterials. The NCLPSI appeared to be replaced with regenerative tissue most rapidly in vivo and scored highest in all subjective evaluations of ease of use. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggested that NCLPSI and CLPSI have favorable properties for further investigation of clinical application in orthopedic tissue engineering.

Recovery of function in skeletal muscle following 2 different contraction-induced injuries.

OBJECTIVE: To determine if the proliferation of myogenic cells is equally important to recovery of contractile function after 2 different types of contraction-induced muscle injuries. DESIGN: Randomized trial. SETTING: Muscle biology laboratory. ANIMALS: Adult male Sprague-Dawley rats. INTERVENTIONS: Tibialis anterior muscles were injured by a single lengthening contraction with large strain (1R) or multiple lengthening contractions with small strain (MR). The hindlimbs of some animals in each group were irradiated before injury to prevent proliferation of myogenic cells during recovery. MAIN OUTCOME MEASURES: Contractile tension was measured immediately after injury and 3, 7, 14, and 21 days after injury. Permeation to Evans blue dye was used to assay membrane damage. Centrally nucleated fibers and reverse transcriptase-polymerase chain reaction of myoD and myogenin were used as measures of myogenesis. RESULTS: Inhibiting myogenesis prevented the recovery of contractile function after MR, but not after 1R. Both protocols caused Evans blue dye uptake immediately after injury, but Evans blue dye was only retained in fibers for several days after 1R. This suggests that membranes reseal after 1R, but not after MR. CONCLUSIONS: The mechanisms that underlie recovery after injuries caused by repeated lengthening contractions and injuries caused by a single lengthening contraction are different. The differences may be important when planning targeted rehabilitation strategies for each type of injury.

The introduction of an extracellular matrix-based scaffold to the marketplace.

Continuous application of new scientific knowledge is a central characteristic of modern medical practice. The current pace of medical innovation creates an environment of rapid change, and the introduction of innovative treatments in the area of regenerative medicine in orthopaedics prompts health care providers, medical device companies, patient advocacy groups, and health insurance payors to study the most optimal method for introducing these treatments to clinical practice. Questions regarding the role and value of preclinical testing, clinical trials, and postmarketing surveillance are pertinent to this discussion, and answers to these questions should culminate in a strategy that benefits patient care.

Bioscaffolds in tissue engineering: a rationale for use in the reconstruction of musculoskeletal soft tissues.

Bioscaffolds derived from animal tissues can be an appealing substrate to induce the formation of functional tissue (histogenesis) within the context of tissue engineering. Bioscaffolds obtained from the extracellular matrix not only contain collagen, which can provide mechanical support, but also include the required biologically active molecules that provide a stimulus for active tissue remodeling. Manufacturing, processing, and the tissue source of the biological scaffold affect the biologic outcome and are important in predicting the clinical results. This article discusses the merits and limitations of using bioscaffolds in soft tissue engineering.

Dexamethasone and recovery of contractile tension after a muscle injury.

Muscle strains, frequently the result of a lengthening contraction, sometimes are treated with corticosteroids. We tested whether an injection of dexamethasone administered soon after muscle injury would minimize inflammation and facilitate the recovery of contractile tension. We applied one eccentric contraction on the tibialis anterior of 76 rats, which were randomly assigned to one of three groups: sham-injured plus dexamethasone, injured plus vehicle, and injured plus dexamethasone. Electrophysiology, histology, and reverse transcription-polymerase chain reaction were used to study the relation between contractile tension, inflammation, and the expression of inflammatory molecules. The single eccentric contraction led to a reversible muscle injury characterized initially by reduced contractile tension and inflammation. The dexamethasone injection reduced the expression of interleukin-1beta and transforming growth factor-beta1 compared with injured vehicle-injected controls and led to a transient improvement of contractile tension 3 days after the injury. No adverse effects were seen for as much as 3 weeks after the dexamethasone injection. The data indicate that one dose of dexamethasone administered soon after muscle strain may facilitate recovery of contractile tension without causing major adverse consequences in this experimental model.

The contribution of contractile pre-activation to loss of function after a single lengthening contraction.

PURPOSE: Some muscle injuries are the result of a single lengthening contraction. Our goal was to evaluate the contributions of angular velocity, arc of motion, and timing of contractile activation relative to the onset of joint motion in an animal model of muscle injury using a single lengthening contraction. METHODS: The intact tibialis anterior (TA) muscle of rats was activated while lengthened, preceded by a maximal isometric contraction of 0, 25, 50, 100, or 200 ms. The lengthening contraction was performed at two different angular velocities (300 or 900 degrees/s) and through two different arcs of motion (90 degrees or 45 degrees). RESULTS: Muscle contractile function, as measured by maximal isometric tetanic tension, was significantly decreased only when the TA was activated at least 50 ms prior to the motion, regardless of angular velocity or arc of motion. CONCLUSION: The data indicated that the duration of an isometric contraction prior to a single lengthening contraction determined the extent of muscle injury irrespective of two different angular velocities.

Muscle molecular phenotype after stroke is associated with gait speed.

The disability of patients after stroke is generally attributed to upper motor neuron defects, but secondary changes in paretic muscle may enhance the disability. We analyzed the molecular phenotype and metabolic profile of the paretic and nonparetic vastus lateralis (VL) and we measured the severity of gait deficit in 13 patients at least 6 months after ischemic stroke. The results showed a significant increase in the proportion of fast myosin heavy chain (MHC, 68 +/- 14%) in the paretic compared to the nonparetic VL (50 +/- 13%). The specific activity of citrate synthase and glyceraldehyde phosphodehydrogenase was not significantly different between the two sides. The proportion of fast MHC was inversely associated with severity of gait deficit indexed by self-selected walking speed in the paretic leg, but not the nonparetic leg. Our findings demonstrate significant and potentially modifiable secondary biologic changes in hemiparetic muscle phenotype that may contribute to the disability of stroke.

Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury.

The purpose of this study was to evaluate the integrity of the muscle membrane and its associated cytoskeleton after a contraction-induced injury. A single eccentric contraction was performed in vivo on the tibialis anterior (TA) of male Sprague-Dawley rats at 900 degrees /s throughout a 90 degrees -arc of motion. Maximal tetanic tension (Po) of the TAs was assessed immediately and at 3, 7, and 21 days after the injury. To evaluate sarcolemmal integrity, we used an Evans blue dye (EBD) assay, and to assess structural changes, we used immunofluorescent labeling with antibodies against contractile (myosin, actin), cytoskeletal (alpha-actinin, desmin, dystrophin, beta-spectrin), integral membrane (alpha- and beta-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P0 was significantly reduced to 4.23 +/- 0.22 N, compared with 8.24 +/- 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 +/- 22% of fibers from the injured TA, compared with 0% in noninjured controls. We found a significant association between EBD-positive fibers and the loss of complete dystrophin labeling. The loss of dystrophin was notable because organization of other components of the subsarcolemmal cytoskeleton was affected minimally (beta-spectrin) or not at all (alpha- and beta-dystroglycan). Labeling with specific antibodies indicated that dystrophin's COOH terminus was selectively more affected than its rod domain. Twenty-one days after injury, contractile properties were normal, fibers did not contain EBD, and dystrophin organization and protein level returned to normal. These data indicate the selective vulnerability of dystrophin after a single eccentric contraction-induced injury and suggest a critical role of dystrophin in force transduction.

Biolistic transfection of cultured myotubes.

Transfection of cells in culture with cDNA constructs is a powerful tool in cell biology, but postmitotic cells, including myotubes, can be hard to transfect with classic methods. Biolistics provides an alternative. We have used this biolistic technique to introduce cDNAs into cultured rat, chick, and C2C12 myotubes. This protocol results in efficient (20 to 70%, depending on cell type) transfection of myotubes, high levels of cDNA expression in individual myotubes, and little cellular damage. Using this procedure, we have expressed different muscle-specific cDNAs as green fluorescent protein (GFP) fusions. This technique is rapid, reliable, uses minimal amounts of reagent per transfection, and yields high transfection rates in a previously hard-to-transfect cell type. Its efficiency and reliability are high, regardless of plasmid size or epitope tag. Muscle cell biologists may now perform experiments in mature myotubes rather than relying on transfection of myoblast cultures or heterologous expression systems.

Lengthening of muscle during distraction osteogenesis.

Chronic lengthening of immobilized, neurally intact muscle leads to the addition of sarcomeres in series. Confirmation of a similar adaptation during distraction osteogenesis is crucial for providing a rationale for a successful outcome of the intervention. When distraction osteogenesis (at < or = 1.4 mm/day) is done in skeletally immature animals, muscle adapts by creating a longer and functionally intact muscle. This is achieved through muscle growth, the proliferation of myogenic cells ultimately leading to serial addition of sarcomeres. When distraction osteogenesis is done in skeletally mature animals, however, the same distraction regimen leads to a lengthened muscle that has significant fibrosis and weakness, the latter possibly a result of partial denervation. Despite a modest but significant elevation of local insulinlike growth factor-1 in the lengthened muscles from adult animals, muscle growth is not adequate and leads to a loss of function. In adult animals, the distraction osteogenesis-induced increase in insulinlike growth factor-1 is insufficient to facilitate muscle growth during lengthening. Muscle can be targeted for future therapeutic use of insulinlike growth factor-1; however, such a therapy also may lead to increased fibrosis.