The viable bioengineered allogeneic cellularized construct StrataGraft® synthesizes, deposits, and organizes human extracellular matrix proteins into tissue type-specific structures and secretes soluble factors associated with wound healing.

BACKGROUND: StrataGraft® (allogeneic cultured keratinocytes and dermal fibroblasts in murine collagen-dsat) is an FDA-approved viable bioengineered allogeneic cellularized construct for adult patients with deep partial-thickness burns requiring surgery. We characterized the structural and functional properties of StrataGraft to improve product understanding by evaluating extracellular matrix (ECM) molecule distribution and secreted protein factor expression in vitro. METHODS: ECM protein expression was determined using indirect immunofluorescence on construct cross sections using commercial antibodies against collagen III, IV, VI, laminin-332, and decorin. Human collagen I expression was verified by enzyme-linked immunosorbent assay (ELISA) for collagen I C-terminal propeptide. Soluble protein factor secretion was quantified by multiplex biomarker assays and singleplex ELISA in conditioned media from meshed constructs. RESULTS: StrataGraft cellular components produced collagen I, collagen III, collagen VI, and decorin in patterns indicating an organized ECM. Distributions of collagen IV and laminin-332 indicated formation of basement membranes and dermal-epidermal junctions. Soluble protein factors were observed in the pg/cm2/h range from 1 h to the experiment end at 168 h. CONCLUSIONS: The organization of the ECM proteins was like human skin and the viable cellular components provided sustained secretion of soluble wound healing factors, making StrataGraft an attractive option for treating severe burns.

Asymmetrical strain distributions and neutral axis location of cartilage in flexure.

Flexural deformation has been used for the biomechanical characterization of native and engineered cartilage and as a mechanical stimulus to induce alteration of cartilage shape during in vitro culture. Flexure is also a physiologically relevant mode of deformation for various cartilaginous structures such as the ears and nose, but a kinematic description of cartilage in flexure is lacking even for simple deformations. The hypothesis of this study was that tension-compression (T-C) nonlinearity of cartilage will result in asymmetrical strain distributions during bending, while a material with similar behavior in tension and compression, such as alginate, will have a more symmetrical distribution of strains. Strips of calf articular cartilage and alginate were tested under uniform circular bending, and strains were determined by a micromechanical analysis of images acquired by epifluorescence microscopy. This experimental analysis was interpreted in the context of a model of small-deflection, pure bending of thin, homogeneous beams of a bimodular elastic material. The results supported the hypothesis and showed that marked asymmetry existed in cartilage flexural strains where the location of the neutral axis was significantly different than the midline and closer to the tensile surface. In contrast, alginate samples had a centrally located neutral axis. These experimental results were supported by the model indicating that the bimodular simplification of cartilage properties is a useful first approximation of T-C nonlinearity in these tests. The neutral axis location in cartilage samples was not influenced by the testing orientation (towards or away from the superficial-most tissue) or magnitude of flexure. These findings characterize the kinematics of cartilage at equilibrium during simple bending and indicate that T-C nonlinearity is an important determinant of the flexural strain distributions in the tested tissue.

The effects of focal articular defects on cartilage contact mechanics.

Focal damage to articular cartilage is common in arthroscopy patients, and may contribute to progressive tissue degeneration by altering the local mechanical environment. The effects of a focal defect, which may be oriented at various orientations relative to the subchondral bone, on the dynamics of cartilage contact and deformation are unclear. The objective of this study was to elucidate the effect of experimental full thickness focal defects, oriented at 80 degrees or 100 degrees relative to the subchondral bone, on intratissue strain and surface sliding of opposing cartilage surfaces during compression and stress relaxation. Pairs of intact bovine osteochondral blocks were compressed uniaxially by 20%, and allowed to stress relax. Tissue deformation was recorded by video microscopy. A full-thickness defect (with either 80 degrees or 100 degrees edges) was created in one block from each pair. Blocks were allowed to reswell and retested. Defect edges were then recut with the opposite orientation, allowed to reswell, and retested again. Stained nuclei were tracked by digital image correlation and used to quantify cartilage strains and surface sliding. The results indicated that loading of intact samples caused axial strain magnitudes that decreased with depth and relatively little sliding. With loading of samples containing defects, strain magnitudes were elevated in cartilage adjacent to, and opposing, defects. For samples with edge orientations of 100 degrees, sliding magnitudes were increased over surfaces adjacent to defects. These local mechanical changes due to full-thickness articular cartilage defects may contribute to altered chondrocyte metabolism, tissue damage, or accelerated wear.

The effects of focal articular defects on intra-tissue strains in the surrounding and opposing cartilage.

Focal damage to articular cartilage is commonly found in symptomatic knees and may contribute to patient discomfort and progressive cartilage degeneration. The objective of this study was to quantify changes in cartilage intra-tissue strain and sliding occurring near a focal defect. Pairs of human osteochondral blocks were compressed by 20% of the total cartilage thicknesses, and tissue deformation was recorded by video microscopy. Then, a single, full-thickness defect was created in one block from each pair, blocks were allowed to re-swell, and the pairs were retested. Stained nuclei, acting as fiducial markers, were tracked by digital image correlation and used to calculate cartilage strains and surface displacement. With intact samples, axial strain decreased with depth, as is typical of cartilage, and relatively little sliding occurred between surfaces. With defect samples, axial compression of cartilage at the defect rim rose by approximately 30%, shear in the opposing tissue increased 10-fold to approximately 0.15, and local sliding was elevated to > 50 microm. In vivo, tissue near a defect likely experiences increased overall compression, magnifying these observed in vitro effects. Excessive strains may contribute to cell death, matrix damage, or accelerated wear, and repair efficacy may depend on the ability to alleviate adverse mechanical conditions.

Shear deformation kinematics during cartilage articulation: effect of lubrication, degeneration, and stress relaxation.

During joint articulation, the biomechanical behavior of cartilage not only facilitates load-bearing and low-friction, but also provides regulatory cues to chondrocytes. Elucidation of cartilage kinematics under combined compression and shearing conditions clarifies these cues in health and disease. The objectives of this study were to elucidate the effects of lubricant, tissue degeneration, and stress relaxation duration on cartilage shear kinematics during articulation. Human osteochondral cores with normal and mildly degenerate surface structures were isolated. Paired blocks from each core were apposed, compressed, allowed to stress relax for 5 or 60 min, and shear tested with a micro-scale video microscopy system using phosphate-buffered saline (PBS) or synovial fluid as lubricant. During applied lateral motion, local and overall shear strain (Exz) of articular cartilage were determined. The applied lateral displacement at which Exz reached 50% of the peak (Deltax(1/2)) was also determined. Quantitatively, surface Exz increased at the onset of lateral motion and peaked just as surfaces detached and slid. With continued lateral motion, surface Exz was maintained. After short stress relaxation, effects of lubrication on Exz and Deltax(1/2) were not apparent. With prolonged stress relaxation, Exz and Deltax(1/2) near the articular surface increased markedly when PBS was used as lubricant. Similar patterns were observed for overall Exz and Deltax(1/2). With degeneration, surface Exz was consistently higher for all cases after the onset of lateral motion. Thus, cartilage shear kinematics is markedly affected by lubricant, cartilage degeneration, and loading duration. Changes in these factors may be involved in the pathogenesis of osteoarthritis.

Biomechanics of cartilage articulation: effects of lubrication and degeneration on shear deformation.

OBJECTIVE: To characterize cartilage shear strain during articulation, and the effects of lubrication and degeneration. METHODS: Human osteochondral cores from lateral femoral condyles, characterized as normal or mildly degenerated based on surface structure, were selected. Under video microscopy, pairs of osteochondral blocks from each core were apposed, compressed 15%, and subjected to relative lateral motion with synovial fluid (SF) or phosphate buffered saline (PBS) as lubricant. When cartilage surfaces began to slide steadily, shear strain (Exz) and modulus (G) overall in the full tissue thickness and also as a function of depth from the surface were determined. RESULTS: In normal tissue with SF as lubricant, Exz was highest (0.056) near the articular surface and diminished monotonically with depth, with an overall average Exz of 0.028. In degenerated cartilage with SF as lubricant, Exz near the surface (0.28) was 5-fold that of normal cartilage and localized there, with an overall E(xz) of 0.041. With PBS as lubricant, Exz values near the articular surface were approximately 50% higher than those observed with SF, and overall Exz was 0.045 and 0.062 in normal and degenerated tissue, respectively. Near the articular surface, G was lower with degeneration (0.06 MPa, versus 0.18 MPa in normal cartilage). In both normal and degenerated cartilage, G increased with tissue depth to 3-4 MPa, with an overall G of 0.26-0.32 MPa. CONCLUSION: During articulation, peak cartilage shear is highest near the articular surface and decreases markedly with depth. With degeneration and diminished lubrication, the markedly increased cartilage shear near the articular surface may contribute to progressive cartilage deterioration and osteoarthritis.

Experimental measurement and quantification of frictional contact between biological surfaces experiencing large deformation and slip.

Interactions between contacting biological surfaces may play significant roles in physiological and pathological processes. Theoretical models have described some special cases of contact, using one or more simplifying assumptions. Experimental quantification of contact could help to validate theoretical analyses. The objective of this study was to develop a general mathematical approach describing the dynamics of deformation and relative surface motion between contacting bodies and to implement this approach to describe the contact between two experimentally tracked tissue surfaces. A theoretical formulation (in 2-D and 3-D) of contact using the movement of discrete tissue markers is described. The method was validated using theoretically generated 3-D datasets, with <1% error for a wide range of parameters. The method was applied to the contact loading of opposing articular cartilage tissues, where displacements of cell nuclei were tracked optically and used to quantify the movements and deformations of the surfaces. Compared to tissues with matched material properties, tissues with mismatched material properties exhibited increased disparities in lateral expansion and relative motion (sliding) between the contacting surfaces.

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage.

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.

Mechanical characterization of native and tissue-engineered cartilage.

Cartilage functions as a low-friction, wear-resistant, load-bearing tissue. During a normal gait cycle, one cartilage surface rolls and slides against another, all the while being loaded and unloaded. The durability of the tissue also makes for an impressive material to study. However, when cartilage is damaged or diseased, the tissue has little capacity to repair itself. The goal of cell-based repair strategies to replace damaged or diseased tissue requires that the functional biomechanical properties of normal (developing or mature), diseased, and repair cartilage be restored. This chapter addresses some of the major methods used to assess the biomechanical properties of native and tissue-engineered cartilage. First, the traditional methods of testing by compression, tension, shear, and indentation are reviewed. Next, additional methods to evaluate interfacial mechanics and lubrication are described. Thus, a variety of mechanical tests may be used to assess functional properties for normal, diseased, and tissue-engineered cartilage.