Myron Spector

Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes

This study directly compared the behaviour of chondrocytes in porous matrices comprising different collagen types and different pore diameters. There was a dramatic difference in the morphology of the cells in the type I and type II collagen matrices. The cells in the type II collagen matrix retained their chondrocytic morphology and synthesized glycosaminoglycans, while in the type I matrix the chondrocytes displayed a fibroblastic morphology with less biosynthetic activity than those in the type II. Small pore diameter affected morphology initially in the type I matrices and showed a higher increase of DNA content, but with time the cells lost the chondrocytic morphology. Our results demonstrate the marked influence of collagen type and pore characteristics on the phenotypic expression of seeded chondrocytes.

Effect of Cultured Autologous Chondrocytes on Repair of Chondral Defects in a Canine Model

Articular cartilage has a limited capacity for repair. In recent clinical and animal experiments, investigators have attempted to elicit the repair of defects of articular cartilage by injecting cultured autologous chondrocytes under a periosteal flap (a layer of periosteum). The objective of the present study was to determine the effect of cultured autologous chondrocytes on healing in an adult canine model with use of histomorphometric methods to assess the degree of repair. A total of forty-four four-millimeter-diameter circular defects were created down to the zone of calcified cartilage in the articular cartilage of the trochlear groove of the distal part of the femur in fourteen dogs. The morphology and characteristics of the original defects were defined in an additional six freshly created defects in three other dogs. Some residual non-calcified articular cartilage, occupying approximately 2 per cent of the total cross-sectional area of the defect, was sometimes left in the defect. The procedure sometimes damaged the calcified cartilage, resulting in occasional microfractures or larger fractures, thinning of the zone of calcified cartilage, or, rarely, small localized penetrations into subchondral bone. The forty-four defects were divided into three treatment groups. In one group, cultured autologous chondrocytes were implanted under a periosteal flap. In the second group, the defect was covered with a periosteal flap but no autologous chondrocytes were implanted. In the third group (the control group), the defects were left empty. The defects were analyzed after twelve or eighteen months of healing. Histomorphometric measurements were made of the percentage of the total area of the defect that became filled with repair tissue, the types of tissue that filled the defect, and the integration of the repair tissue with the adjacent cartilage at the sides of the defects and with the calcified cartilage at the base of the defect. In histological sections made through the center of the defects in the three groups, the area of the defect that filled with new repair tissue ranged from a mean total value of 36 to 76 per cent, with 10 to 23 per cent of the total area consisting of hyaline cartilage. Integration of the repair tissue with the adjacent cartilage at the edges of the defect ranged from 16 to 32 per cent in the three groups. Bonding between the repair tissue and the calcified cartilage at the base of the defect ranged from 41 to 89 per cent. With the numbers available, we could detect no significant difference among the three groups with regard to any of the parameters used to assess the quality of the repair. In the two groups in which a periosteal flap was sutured to the articular cartilage surrounding the defect, the articular cartilage showed degenerative changes that appeared to be related to that suturing. CLINICAL RELEVANCE: The technique of injecting cultured autologous chondrocytes under a periosteal flap recently was introduced to treat defects in the articular cartilage of humans. The long-term efficacy of this treatment is unknown. An animal model was developed to evaluate the procedure and its effectiveness.

Histologic analysis of tissue after failed cartilage repair procedures.

This study evaluated the composition of reparative tissue retrieved during revision surgery from full thickness chondral defects in 18 patients in whom abrasion arthroplasty (n = 12), grafting of perichondrial flaps (n = 4), and periosteal patching augmented by autologous chondrocyte implantation in cell suspension (n = 6) failed to provide lasting relief of symptoms. The defects were graded by gross appearance, and all of the tissue filling the defect was retrieved. Histologic evaluation included histomorphometric analysis of the percentage of selected tissue types in cross sections. Immunohistochemistry was performed using antibodies to Types I, II, and X collagen. The histologic appearance of material retrieved after abrasion arthroplasty was that of fibrous, spongiform tissue comprising Type I collagen in 22% +/- 9% (mean +/- standard error of the mean) of the cross sectional area, and degenerating hyaline tissue (30% +/- 10%) and fibrocartilage (28% +/- 7%) with positive Type II collagen staining. Three of four specimens obtained after implantation of perichondrium failed as a result of bone formation that was found in 19% +/- 6% of the cross sectional area, including areas staining positive for Type X collagen, as an indicator for hypertrophic chondrocytes. Revision after autologous chondrocyte implantation was associated with partial displacement of the periosteal graft from the defect site because of insufficient ongrowth or early suture failure. When the graft edge displaced, repair tissue was fibrous (55% +/- 11%), whereas graft tissue attached to subchondral bone displayed hyaline tissue (to 6%) and fibrocartilage (to 12%) comprising Type II collagen at 3 months after surgery. Evaluation of retrieved repair tissue after selected cartilage repair procedures revealed distinctive histologic features reflecting the mechanisms of failure.

The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis

The healing of articular cartilage defects may be improved by the use of implantable three-dimensional matrices. The present study investigated the effects of four cross-linking methods on the compressive stiffness of collagen-glycosaminoglycan (CG) matrices and the interaction between adult canine articular chondrocytes and the matrix: dehydrothermal treatment (DHT), ultraviolet irradiation (UV), glutaraldehyde treatment (GTA), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The degree and kinetics of chondrocyte-mediated contraction, chondrocyte proliferation, and protein and glycosaminoglycan synthesis were evaluated over a four-week period in vitro. Cell-mediated contraction of the matrices varied with cross-linking: the most compliant DHT and UV matrices contracted the most (60% reduction in matrix diameter) and stiffest EDAC matrices contracted the least (30% reduction in matrix diameter). All cross-linking protocols permitted cell proliferation and matrix synthesis as measured by DNA content and radiolabeled sulfate and proline incorporation, respectively. During the first week in culture, a lower level of proliferation was seen in the GTA matrices but over the four-week culture period, the GTA and EDAC matrices provided for the greatest cell proliferation. On day 2, there was a significantly lower rate of 3H-proline incorporation in the GTA matrices (p<0.003) although at later time points, the EDAC and GTA matrices exhibited the highest levels of matrix synthesis. With regard to cartilage-specific matrix molecule synthesis, immunohistochemistry revealed a greater amount of type II collagen in DHT and UV matrices at the early time points. These findings serve as a foundation for future studies of tissue engineering of articular cartilage and the association of chondrocyte contraction and the processes of mitosis and biosynthesis.

Canine chondrocytes seeded in type I and type II collagen implants investigated In Vitro

Synthetic and natural absorbable polymers have been used as vehicles for implantation of cells into cartilage defects to promote regeneration of the articular joint surface. Implants should provide a pore structure that allows cell adhesion and growth, and not provoke inflammation or toxicity when implanted in vivo. The scaffold should be absorbable and the degradation should match the rate of tissue regeneration. To facilitate cartilage repair the chemical structure and pore architecture of the matrix should allow the seeded cells to maintain the chondrocytic phenotype, characterized by synthesis of cartilage-specific proteins. We investigated the behavior of canine chondrocytes in two spongelike matrices in vitro: a collagen-glycosaminoglycan (GAG) copolymer produced from bovine hide consisting of type I collagen and a porous scaffold made of type II collagen by extraction of porcine cartilage. Canine chondrocytes were seeded on both types of matrices and cultured for 3 h, 7 days, and 14 days. The histology of chondrocyte-seeded implants showed a significantly higher percentage of cells with spherical morphology, consistent with chondrocytic morphology, in the type II sponge at each time point. Pericellular matrix stained for proteoglycans and for type II collagen after 14 days. Biochemical analysis of the cell seeded sponges for GAG and DNA content showed increases with time. At day 14 there was a significantly higher amount of DNA and GAG in the type II matrix. This is the first study that directly compares the behavior of chondrocytes in type I and type II collagen matrices. The type II matrix may be of value as a vehicle for chondrocyte implantation on the basis of the higher percentage of chondrocytes retaining spherical morphology and greater biosynthetic activity that was reflected in the greater increase of GAG content.

Histological changes in the human anterior cruciate ligament after rupture.

Background: Four phases in the response to injury of the ruptured human anterior cruciate ligament are observed histologically; these include an inflammatory phase, an epiligamentous repair phase, a proliferative phase, and a remodeling phase. One objective of this study was to describe the histological changes that occur in the ruptured human anterior cruciate ligament during these phases. Myofibroblast-like cells that contain a-smooth muscle actin are present in the midsubstance of the intact human anterior cruciate ligament. A second objective of this study was to determine whether an increased number of myofibroblast-like cells is found in the midsubstance of the ruptured human anterior cruciate ligament because it was thought that those cells might be responsible in part for the retraction of the ruptured anterior cruciate ligament. In the early phase of this study, it was found that the number of myofibroblast-like cells in the midsubstance of the ruptured anterior cruciate ligament was actually decreased, and this hypothesis was abandoned. During the epiligamentous repair phase, synovial tissue was formed that covered the ends of the ruptured anterior cruciate ligament. Most of the synovial lining cells were myofibroblast-like cells that contained a-smooth muscle actin. The primary objective of this study was to determine the location and the characteristics of the a-smooth muscle actin-containing myofibroblast-like cells that appear in the human anterior cruciate ligament following rupture. Methods: Twenty-three ruptured and ten intact human anterior cruciate ligaments were evaluated for cellularity, nuclear morphology, blood vessel density, and percentage of cells containing a contractile actin isoform, a-smooth muscle actin. The histological features of the synovial and epiligamentous tissues were also described. Results: At no time after rupture was there evidence of tissue-bridging between the femoral and tibial remnants of the anterior cruciate ligament. The ruptured ligaments demonstrated a time-dependent histological response, which consisted of inflammatory cell infiltration up to three weeks, gradual epiligamentous repair and resynovialization between three and eight weeks, and neovascularization and an increase in cell number density between eight and twenty weeks. Compared with the intact ligaments, there was a decrease in the percentage of myofibroblast-like cells containing a-smooth muscle actin within the remnant of the ligament. However, many of the epiligamentous and synovial cells encapsulating the remnants contained a-smooth muscle actin. Conclusions: After rupture, the human anterior cruciate ligament undergoes four histological phases, consisting of inflammation, epiligamentous regeneration, proliferation, and remodeling. The response to injury is similar to that reported in other dense connective tissues, with three exceptions: formation of an a-smooth muscle actin-expressing synovial cell layer on the surface of the ruptured ends, the lack of any tissue bridging the rupture site, and the presence of an epiligamentous reparative phase that lasts eight to twelve weeks. Other characteristics reported in healing dense connective tissue, such as fibroblast proliferation, expression of a-smooth muscle actin, and revascularization, also occur in the ruptured human anterior cruciate ligament. Clinical Relevance: Unlike extra-articular ligaments that heal after injury, the human intra-articular anterior cruciate ligament forms a layer of synovial tissue over the ruptured surface, which may impede repair of the ligament. Moreover, a large number of cells in this synovial layer and in the epiligamentous tissue express the gene for a contractile actin isoform, a-smooth muscle actin, thus differentiating into myofibroblasts. These events may play a role in the retraction and lack of healing of the ruptured anterior cruciate ligament.

Collagen-GAG substrate enhances the quality of nerve regeneration through collagen tubes up to level of autograft.

Peripheral nerve regeneration was studied across a tubulated 10-mm gap in the rat sciatic nerve using histomorphometry and electrophysiological measurements of A-fiber, B-fiber, and C-fiber peaks of the evoked action potentials. Tubes fabricated from large-pore collagen (max. pore diameter, 22 nm), small-pore collagen (max. pore diameter, 4 nm), and silicone were implanted either saline-filled or filled with a highly porous, collagen-glycosaminoglycan (CG) matrix. The CG matrix was deliberately synthesized, based on a previous optimization study, to degrade with a half-life of about 6 weeks and to have a very high specific surface through a combination of high pore volume fraction (0.95) and relatively small average pore diameter (35 microm). Nerves regenerated through tubes fabricated from large-pore collagen and filled with the CG matrix had significantly more large-diameter axons, more total axons, and significantly higher A-fiber conduction velocities than any other tubulated group; and, although lower than normal, their histomorphometric and electrophysiological properties were statistically indistinguishable from those of the autograft control. Although the total number of myelinated axons in nerves regenerated by tubulation had reached a plateau by 30 weeks, the number of axons with diameter larger than 6 microm, which have been uniquely associated with the A-fiber peak of the action potential, continued to increase at substantial rates through the completion of the study (60 weeks). The kinetic data strongly suggest that a nerve trunk maturation process, not previously reported in studies of the tubulated 10-mm gap in the rat sciatic nerve, and consisting in increase of axonal tissue area with decrease in total tissue area, continues beyond 60 weeks after injury, resulting in a nerve trunk which increasingly approaches the structure of the normal control.

Effects of a cultured autologous chondrocyte‐seeded type II collagen scaffold on the healing of a chondral defect in a canine model

Using a previously established canine model for repair of articular cartilage defects, this study evaluated the 15‐week healing of chondral defects (i.e., to the tidemark) implanted with an autologous articular chondrocyte‐seeded type II collagen scaffold that had been cultured in vitro for four weeks prior to implantation. The amount and composition of the reparative tissue were compared to results from our prior studies using the same animal model in which the following groups were analyzed: defects implanted with autologous chondrocyte‐seeded collagen scaffolds that had been cultured in vitro for approximately 12 h prior to implantation, defects implanted with autologous chondrocytes alone, and untreated defects. Chondrocytes, isolated from articular cartilage harvested from the left knee joint of six adult canines, were expanded in number in monolayer for three weeks, seeded into porous type II collagen scaffolds, cultured for an additional four weeks in vitro and then implanted into chondral defects in the trochlear groove of the right knee joints. The percentages of specific tissue types filling the defects were evaluated histomorphometrically and certain mechanical properties of the repair tissue were determined. The reparative tissue filled 88 ± 6% (mean ± SEM; range 70–100%) of the cross‐sectional area of the original defect, with hyaline cartilage accounting for 42 ± 10% (range 7–67%) of defect area. These values were greater than those reported previously for untreated defects and defects implanted with a type II collagen scaffold seeded with autologous chondrocytes within 12 h prior to implantation. Most striking, was the decreased amount of fibrous tissue filling the defects in the current study, 5 ± 5% (range 0–26%) as compared to previous treatments. Despite this improvement, indentation testing of the repair tissue formed in this study revealed that the compressive stiffness of the repair tissue was well below (20‐fold lower stiffness) that of native articular cartilage.

A self-powered mechanical strain energy sensor

With the growing use of sensors in various structural and mechanical systems, the powering and communication of these sensors will become a critical factor. Wireless communication electronics are becoming ubiquitous and with the decreasing electrical power requirements for these circuits it is now feasible to generate power from the conversion of mechanical energy into electrical energy. This paper focuses on the theoretical and experimental analysis of a simple mechanical strain energy sensor with wireless communication. A simple beam bending experiment is given to illustrate some of the characteristics of the self-powered strain energy sensor.

Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment

An injectable and biodegradable hydrogel system comprising hyaluronic acid-tyramine (HA-Tyr) conjugates can safely undergo covalent cross-linking in vivo by the addition of small amounts of peroxidase and hydrogen peroxide (H(2)O(2)), with the independent tuning of the gelation rate and degree of cross-linking. Such hydrogel networks with tunable mechanical and degradation properties may provide the additional level of control needed to enhance chondrogenesis and overall cartilage tissue formation in vitro and in vivo. In this study, HA-Tyr hydrogels were explored as biomimetic matrices for caprine mesenchymal stem cells (MSCs) in cartilage tissue engineering. The compressive modulus, equilibrium swelling and degradation rate could be controlled by varying the concentration of H(2)O(2) as the oxidant in the oxidative coupling reaction. Cellular condensation reflected by the increase in effective number density of rounded cells in lacunae was greater in softer hydrogel matrices with lower cross-linking that displayed enhanced scaffold contracture. Conversely, within higher cross-linked matrices, cells adopted a more elongated morphology, with a reduced degree of cellular condensation. Furthermore, the degree of hydrogel cross-linking also modulated matrix biosynthesis and cartilage tissue histogenesis. Lower cross-linked matrix enhanced chondrogenesis with increases in the percentage of cells with chondrocytic morphology; biosynthetic rates of glycosaminoglycan and type II collagen; and hyaline cartilage tissue formation. With increasing cross-linking degree and matrix stiffness, a shift in MSC differentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tissues was observed. These findings suggest that the tunable three-dimensional microenvironment of the HA-Tyr hydrogels modulates cellular condensation during chondrogenesis and has a dramatic impact on spatial organization of cells, matrix biosynthesis, and overall cartilage tissue histogenesis.