Hu-Ping Hsu

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.

Effect of knee component alignment on tibial load distribution with clinical correlation

To determine ideal alignment and component placement of total knee prostheses, Kinematic (K) and total condylar (TC) devices were physiologically loaded and interface forces were measured. Laboratory observations were correlated with clinical (roentgenographic) findings. Asymmetric loading of the tibial component has been proposed as causing loosening and radiolucent lines. Misalignment of components is one factor that affects load sharing by bone under the medial and lateral regions of the tibial plateau. Tibial components of K and TC prostheses were inserted without cement into the cut surfaces of artificial tibiae. The mating femoral condylar components were mounted. The tibial and femoral components were individually positioned at 0 degrees (horizontal) and at certain angles of varus and valgus. Pressure-sensitive film was placed between the tibial component and the artificial tibia. A vertical load of 1500 N was used. The experiment was replicated twice. The percentages of the load on the medial and lateral regions of the tibial plateau were calculated from quantitative image analysis of the pressure patterns on the film. Roentgenograms from 532 K and 21 TC patients were examined to determine the orientations of the condylar and tibial components and the presence of radiolucent lines around the tibial component. An even distribution (ideal alignment) of load on the medial and lateral regions of the K tibial component occurred at 9 degrees of valgus tilt of the femoral component and 2 degrees of varus tilt of the tibial component and for the TC at 7 degrees valgus and 0 degrees varus. Misalignment by 5 degrees yielded a 7% change in the load distribution under the K plateau and a 40% change for the TC prosthesis; a 10 degrees misalignment produced changes of 34% and 62% for the K and TC, respectively. Small variations in clinical knee alignment produced the same percentage of radiolucent lines for each alignment group. The location of radiolucent lines was distributed among the medial, lateral, and both tibial plateaus regardless of knee alignment, although there were more medial reactions overall. The smallest incidence (8%) of radiolucent lines occurred with the K prosthesis at 7 degrees of knee valgus, the femoral component placed at 9 degrees valgus, and the tibial component at 2 degrees varus. This correlated with the ideal bench-test findings for the K device.

The Effectiveness of the Controlled Release of Gentamicin from Polyelectrolyte Multilayers in the Treatment of Staphylococcus aureus Infection in a Rabbit Bone Model

While the infection rate of orthopedic implants is low, the required treatment, which can involve six weeks of antibiotic therapy and two additional surgical operations, is life threatening and expensive, and thus motivates the development of a one-stage re-implantation procedure. Polyelectrolyte multilayers incorporating gentamicin were fabricated using the layer-by-layer deposition process for use as a device coating to address an existing bone infection in a direct implant exchange operation. The films eluted about 70% of their payload in vitro during the first three days and subsequently continued to release drug for more than four additional weeks, reaching a total average release of over 550 microg/cm(2). The coatings were demonstrated to be bactericidal against Staphylococcus aureus, and degradation products were generally nontoxic towards MC3T3-E1 murine preosteoblasts. Film-coated titanium implants were compared to uncoated implants in an in vivo S. aureus bone infection model. After a direct exchange procedure, the antimicrobial-coated devices yielded bone homogenates with a significantly lower degree of infection than uncoated devices at both day four (p < 0.004) and day seven (p < 0.03). This study has demonstrated that a self-assembled ultrathin film coating is capable of effectively treating an experimental bone infection in vivo and lays the foundation for development of a multi-therapeutic film for optimized, synergistic treatment of pain, infection, and osteomyelitis.

Connective tissue response to tubular implants for peripheral nerve regeneration: The role of myofibroblasts

The presence of contractile cells, their organization around regenerating nerve trunks, and the hypothetical effect of these organized structures on the extent of regeneration across a tubulated 10‐mm gap in the rat sciatic nerve were investigated. Collagen and silicone tubes were implanted both empty and filled with a collagen‐glycosaminoglycan (GAG) matrix. Nerves were retrieved at 6, 30, and 60 weeks postoperatively and time‐dependent values of the nerve trunk diameter along the tubulated length were recorded. The presence of myofibroblasts was identified immunohistochemically using a monoclonal antibody to α‐smooth muscle actin. Myofibroblasts were circumferentially arranged around the perimeter of regenerated nerve trunks, forming a capsule which was about 10 times thicker in silicone tubes than in collagen tubes. The nerve trunk diameter that formed inside collagen tubes was twice as large as that inside silicone tubes. In contrast, the collagen‐GAG matrix had a relatively small effect on capsule thickness or diameter of regenerate. It was hypothesized that the frequency of successful bridging by axons depends on the balance between two competitive forces: the axial forces generated by the outgrowth of axons and nonneuronal cells from the proximal stump and the constrictive, circumferential forces imposed by the contractile tissue capsule that promote closure of the wounded stumps and prevent axon elongation. Because the presence of the collagen‐GAG matrix has enhanced greatly the recovery of normal function of regenerates in silicone tubes, it was hypothesized that it accelerated axonal elongation sufficiently before the hypothetical forces constricting the nerve trunk in silicone tubes became sufficiently large. The combined data suggest a new mechanism for peripheral nerve regeneration along a tubulated gap. J. Comp. Neurol. 417:415–430, 2000.

Lubricin distribution in the goat infraspinatus tendon: a basis for interfascicular lubrication.

BACKGROUND This study was motivated by the need to better understand the tribology of the rotator cuff, as it could provide insights into degenerative processes and suggest therapeutic approaches to cuff disorders. The objective was to evaluate the distribution of a known lubricating protein in the infraspinatus tendon of the rotator cuff in adult goats. The hypothesis was that lubricin, also known as superficial zone protein or proteoglycan 4, serves as an interfascicular lubricant. METHODS Eight infraspinatus tendons were resected from eight Spanish goats, and five patellar tendons and articular cartilage samples were also resected from five of the goats. Samples were processed for immunolocalization of lubricin with use of a purified monoclonal antibody and for histological analysis with Masson trichrome staining for collagen. The locations of lubricin within the tendon were documented, and measurements were made of the distance to which lubricin was detected in the tendon, relative to the humeral insertion site, and the depth of lubricin staining into the fibrocartilage of the fascicle bordering the humeral joint space. Images from polarized light microscopy were used to measure the fascicle diameter and the crimp length. RESULTS Lubricin was prominent in layers separating the fascicles in the infraspinatus tendon, with occasional intrafascicular staining. The fibrocartilaginous portion of the fascicle bordering the humeral joint space displayed diffuse lubricin staining. The Masson trichrome staining of the collagen in this lubricin-containing fibrocartilage indicated that it was not being tensioned at the time of fixation, whereas the collagen contained in the body of the tendon was under tension. The crimp of the fascicles near the humeral joint side of the tendon displayed a shorter peak-to-peak length than the crimp of the fascicles in the superior region of the tendon. Only the surface of two of the five patellar tendon samples stained for lubricin; there was no staining within these ligaments. CONCLUSIONS The sheaths of the fascicles of the infraspinatus tendon near the bone insertion site contain lubricin, indicating that this lubricating protein may be facilitating interfascicular movement. The fact that the crimp pattern of fascicles changes with location in the tendon provides support for the supposition that fascicles move relative to one another as the tendon is loaded, underscoring the importance of a lubricating protein in the layer separating the fascicles.

The reparative response to cross-linked collagen-based scaffolds in a rat spinal cord gap model

Prior work demonstrated the improvement of peripheral nerve regeneration in gaps implanted with collagen scaffold-filled collagen tubes, compared with nerve autografts, and the promise of such implants for treating gaps in spinal cord injury (SCI) in rats. The objective of this study was to investigate collagen implants alone and incorporating select therapeutic agents in a 5-mm full-resection gap model in the rat spinal cord. Two studies were performed, one with a 6-week time point and one with a 2-week time point. For the 6-week study the groups included: (1) untreated control, (2) dehydrothermally (DHT)-cross-linked collagen scaffold, (3) DHT-cross-linked collagen scaffold seeded with adult rat neural stem cells (NSCs), and (4) DHT-cross-linked collagen scaffold incorporating plasmid encoding glial cell line-derived neurotropic factor (pGDNF). The 2-week study groups were: (1) nontreated control, (2) DHT-cross-linked collagen scaffold; (3) DHT-cross-linked collagen scaffold containing laminin; and (4) carbodiimide-cross-linked collagen scaffold containing laminin. The tissue filling the defect of all groups at 6 weeks was largely composed of fibrous scar; however, the tissue was generally more favorably aligned with the long axis of the spinal cord in all of the treatment groups, but not in the control group. Quantification of the percentage of animals per group containing cystic cavities in the defect showed a trend toward fewer rats with cysts in the groups in which the scaffolds were implanted compared to control. All of the collagen implants were clearly visible and mostly intact after 2 weeks. A band of fibrous tissue filling the control gaps was not seen in the collagen implant groups. In all of the groups there was a narrowing of the spinal canal within the gap as a result of surrounding soft tissue collapse into the defect. The narrowing of the spinal canal occurred to a greater extent in the control and DHT scaffold alone groups compared to the DHT scaffold/laminin and EDAC scaffold/laminin groups. Collagen biomaterials can be useful in the treatment of SCI to: favorably align the reparative tissue with the long axis of the spinal cord; potentially reduce the formation of fluid-filled cysts; serve as a delivery vehicle for NSCs and the gene for GDNF; and impede the collapse of musculature and connective tissue into the defect.

Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen-glycosaminoglycan matrix

Lapine and canine marrow stromal cells were found to contain a contractile actin isoform, alpha-smooth muscle actin (SMA), by immunohistochemistry and Western blot analysis. The SMA was found to be incorporated into stress fibers that were prominently displayed by the cells in monolayer culture. The cell content of this actin isoform increased with passage number. The contractility of SMA-expressing stromal cells was demonstrated by their contraction of collagen-glycosaminoglycan analogs of extracellular matrix into which they were seeded. The demonstration that marrow-derived stromal cells express the SMA gene may explain recent findings of this expression in musculoskeletal connective tissue cells including osteoblasts, chondrocytes, and fibrochondrocytes that may be derived from this mesenchymal stem cell. The implications of these findings for tissue engineering strategies employing marrow stromal cells are also discussed.

A comparative study of uncemented tibial components

The effect of tibial component stem and post design on load distribution and on displacements at the component-bone interface was investigated. Four different configurations were tested with a flat component used as a control. Loading conditions included vertical central, vertical offset (eg, varus/valgus), shear and torque. Both artificial and cadaveric bones were used in the study. Pressure-sensitive film was used to obtain pressure patterns at the interface. Image processing was then used to quantify the load distribution. For shear and torque, relative motion was seen as a smeared pressure pattern. This was calibrated in terms of microns of displacement as a function of image density. The central stemmed and bladed designs performed better than short-pegged designs, in resistance to offset loading. In shear and torque, short pegs close to the component periphery, or a central stem with blades, produced the least interface displacement. The application of this work to the design of components for both press-fit and cemented application is discussed.

Radiographic study of kinematic total knee arthroplasty

One thousand sixty-nine consecutive cemented Kinematic Condylar total knee arthroplasties performed by one group of orthopaedists were studied. The maximum follow-up period was 7 years. Most patients had rheumatoid arthritis or osteoarthritis, and the average patient age was 67 years (range, 12-90 years). Aseptic revisions for loosening were required for only one tibial component and six patella components. Average postoperative flexion was 2.5-107 degrees. The preoperative to postoperative change in range of flexion was not affected by the tilt angle of the tibial component in the sagittal plane. With the use of external alignment guides, the average postoperative alignment was ideal but the standard deviation was high; the standard deviation and the extremes were lower when intramedullary guides were used. There was a 14% incidence of femoral radiolucency and a 30% incidence of tibial radiolucency, which increased only slightly with time. Most radiolucencies on the tibial side were small and restricted to the extreme edges; rarely did radiolucency occur around the central peg. More than one half of the thicker radiolucencies occurred adjacent to wedge-shaped bone defects that were filled with cement.

Extracorporeal shock wave-induced proliferation of periosteal cells

The cambium cells of the periosteum are an important cell source for select tissue engineering/regenerative medicine applications due to their osteogenic and chondrogenic potential. However, the cambium layer is only 2–5 cells thick, which complicates its harvest, and the low cell number limits its suitability for certain applications. Extracorporeal shock waves (ESWs) have been reported to cause periosteal osteogenesis following cambium layer thickening. This study quantified the proliferation of cambium cells in the femur and tibia of adult rats following ESW treatment at two different energy flux densities. Four days after application of ESWs, there was a significant (3‐ to 6‐fold) increase in cambium layer thickness and cell number. Proliferation was seen with an energy flux density as low as 0.15 mJ/mm2. The tibial cambium cells were more proliferative than those of the femur, with the cells closest to the ESW source proliferating the most. Within the thickened periosteum, α‐smooth muscle actin and von Willebrand Factor expression were upregulated, suggesting a vascular role in ESW osteogenesis. Bone formation was seen within the stimulated periosteum at day 4. We propose that non‐invasive ESWs can be used to rapidly stimulate cambium cell proliferation, providing a larger cell population for use as a progenitor cell source for tissue engineering applications, than can normally be provided by periosteum.