Viability of fibroblast-seeded ligament analogs after autogenous implantation.

Fibroblast-seeded collagen scaffolds or ligament analogs are potentially useful for reconstruction of the anterior cruciate ligament of the knee. To provide lasting benefits, the seeded cells must survive implantation within the harsh synovial environment of the knee joint. Our objective was to determine the in vivo fate of autogenous fibroblast-seeded ligament analogs as a function of fibroblast source (anterior cruciate ligament or skin), implantation site (knee joint or subcutaneous space), and time after implantation (1, 2, 4, 6, or 8 weeks). Before implantation, fibroblasts were labeled with PKH26-GL, a fluorescent membrane dye. Immediately after retrieval of the implant, the viability of the labeled seeded cells was assessed under a fluorescent microscope. Viable seeded fibroblasts remained attached to the collagen fibers within the ligament analogs for at least 4 weeks (skin fibroblasts) or 6 weeks (anterior cruciate ligament fibroblasts) after implantation. A larger number of viable seeded cells were consistently observed in the subcutaneous space than in the knee joint. Scaffold resorption prevented observation at the 8-week time period. Fibroblast-seeded ligament analogs remained viable for prolonged periods in the knee joint and therefore have the potential to influence the formation and remodeling of neoligament tissue after reconstruction of the anterior cruciate ligament.

Bone cell behavior on Matrigel-coated Ca/P coatings of varying crystallinities.

Rat calvarial cell mitogenic behavior was investigated on various biomaterials coated with Matrigel, a basement membrane matrix containing growth factors. Low (20-40%) and high (70-90%) crystallinity hydroxyapatite (rHA and cHA), rough titanium (Ti), and tissue culture polystyrene (TP) surfaces were compared. Surface chemistry and calcium resorption of HA coatings, alkaline phosphatase activity (APA), and growth of cells were measured for Matrigel-coated and uncoated surfaces at 2, 7, and 14 days. Gene expression for four noncollagenous bone-related proteins (osteonectin, osteopontin, alkaline phosphatase, and osteocalcin) was also investigated by reverse transcription and polymerase chain reaction up to 28 days. Ca concentration in incubating solutions increased with time for the two types of HA coatings and was always greater for rHA than cHA. Surface chemistry and coating dissolution rates were not affected by the presence of Matrigel or cells throughout the study. APA of cells on the two HA-coated surfaces was comparably enhanced in the presence of Matrigel and was greater than on Ti surfaces. Only HA surfaces showed an increased APA of cells with time in the presence of Matrigel. Cell growth peaked at 7 days and was greatest for cells on the two HA surfaces and without Matrigel. At 14 days, cell growth was comparable on the four surfaces. The presence of HA and Matrigel enhanced cell-specific APA at 14 days. Gene expression for all four proteins investigated showed no differences between surfaces after 7 days. At 2 and 7 days, gene expression was indicative of proliferation for Ti, and of proliferation, differentiation, and mineralization for HA and TP more so without Matrigel. The addition of this matrix significantly enhanced mitogenicity of calvarial cells on HA only after 14 days. Matrigel eliminated differences seen between the two HA coatings. Gene expression was not enhanced or inhibited by the presence of Matrigel.

Impact loading of the lumbar spine during football blocking.

The purpose of our study was to determine the impact force to the lumbar spine when football players hit a blocking sled. We quantified the loads at the L4-5 motion segment throughout the blocking sequence. Five Division I-A college football linemen were subjects for our study. Kinematic data were obtained while the subjects hit a blocking sled instrumented with a force plate. Three plane forces were then calculated from these data. The average impact force measured at the blocking sled was 3013 +/- 598 N. The average peak compression force at the L4-5 motion segment was 8679 +/- 1965 N. The average peak anteroposterior shear force was 3304 +/- 1116 N, and the average peak lateral shear force was 1709 +/- 411 N. The magnitude of the loads on the L4-5 motion segment during football blocking exceed those determined during fatigue studies to cause pathologic changes in both the lumbar disk and the pars interarticularis. These data suggest that the mechanics of repetitive blocking may be responsible for the increased incidence of lumbar spine injury incurred by football linemen.

Development of fibroblast-seeded ligament analogs for ACL reconstruction.

We fabricated "ligament analogs" in vitro by seeding high-strength resorbable collagen fiber scaffolds with intraarticular (anterior cruciate ligament, ACL) or extraarticular (patellar tendon, PT) rabbit fibroblasts. Fibroblasts attached, proliferated, and secreted new collagen on the ligament analogs in vitro. Fibroblast function depended on the tissue culture substrate (ligament analog vs. tissue culture plate) and the origin of the fibroblasts (ACL vs. PT) PT fibroblasts proliferated more rapidly than ACL fibroblasts when cultured on ligament analogs. Collagen synthesis by ACL and PT fibroblasts was approximately tenfold greater on ligament analogs than on tissue culture plates. The composition, structure, and geometry of the collagen fiber scaffolds may promote collagen synthesis within ligament analogs in vitro. Ligament analogs roughly approximate the structure and strength of native ligament tissue. Ongoing in vivo studies suggest that autogenous fibroblast-seeded ligament analogs remain viable after implantation into the knee joint. With further development, ligament analogs may be useful as implants for ACL reconstruction surgery.

Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment.

The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. We compared the effects of two physical crosslinking methods, ultraviolet irradiation (UV) (254 nm) and dehydrothermal (DHT) treatment, on the mechanical properties and molecular integrity of collagen fibers extruded from an acidic dispersion of type I bovine dermal collagen. Collagen fibers exposed to UV irradiation for 15 min had ultimate tensile strength (54 MPa) and modulus (184 MPa) values greater than or equivalent to values for fibers crosslinked with DHT treatment for 3 or 5 days. UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen fibers. Characterization of collagen extracted from the crosslinked samples by dilute acetic acid and limited pepsin digestion indicate that both UV and DHT treatments cause fragmentation of at least a portion of the collagen molecules. Partial loss of the native collagen structure may influence attachment migration, and proliferation of cells on collagen fiberbased ligament analogs. These issues are currently being addressed in our laboratory.

Effect of Ca/P coating resorption and surgical fit on the bone/implant interface.

The effect of coating resorption on bone apposition and attachment strength to resorbable hydroxyapatite (HA), nonresorbable HA-coated, and uncoated rough titanium implants was evaluated in interference- and noninterference-fit (gap of 2-3 mm) surgical models 2, 4, and 12 weeks postoperatively. Interference and noninterference fits showed differences in bone bridging. Bone apposition was circumferential around the implants in noninterference fit. Significantly greater bone apposition was seen to nonresorbable HA-coated implants than uncoated and resorbable HA-coated implants at 4 and 12 weeks. Only resorbable HA coatings showed significantly lower bone apposition for noninterference versus interference fit and from 4-12 weeks. At 2 weeks, strengths of bone attachment to resorbable HA-coated implants were greater than the other implants, and decreased to lower values (not significant) than the nonresorbable HA-coated implants at 4 and 12 weeks. Differences in push-out shear strengths between interference- and noninterference-fit surgical models were significant for uncoated implants at 4 weeks, but not for HA-coated implants at any time period. Significant differences were seen between the three implant types only for the noninterference-fit model, where the HA-coated implants showed greater strengths than the uncoated implants (significant at 2 and 4 weeks). This study showed that presence of resorbable or nonresorbable HA coatings is beneficial when a gap of 2-3 mm is present between the implant and the bone. The resorbable HA-coated implants showed greatest strengths at the early time period. At later time periods, resorbable HA-coated implants showed lower bone apposition and attachment strengths than nonresorbable HA coatings.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraosseous incorporation of composite collagen prostheses designed for ligament reconstruction.

Composite collagen prostheses are potentially useful for reconstruction of the anterior cruciate ligament (ACL). We evaluated the intraosseous response to composite collagen prostheses to determine if "biological fixation" could be used to secure the prostheses within surgical bone tunnels. The rate of degradation of the prosthesis and the response of the tissue were evaluated, as a function of collagen crosslinking agent and time, in nonloaded bone tunnels in rabbits. Prostheses were fabricated by the alignment of 200 reconstituted type-I collagen fibers (60 microns in diameter) and the embedding of the fibers within a collagen matrix. The prostheses degraded rapidly within the bone tunnels in comparison with soft-tissue implantation sites. Dehydrothermal-cyanamide crosslinked collagen fibers were completely degraded by 8 weeks. Only 10% of glutaraldehyde cross-linked collagen fibers remained intact at 12 weeks. Fibrous tissue and inflammatory cells rapidly infiltrated the prostheses, and new bone surrounded the circumference of the prostheses, advancing toward the center at longer times. At the lateral cortex, where fibrous tissue emerged, the bone/soft-tissue interface was delineated by a tidemark, similar to that observed in a normal ligament insertion site. Preliminary pull-out testing of the soft tissue from the bone was discontinued because failure consistently occurred in the soft tissue; this suggests rapid incorporation of the prostheses within the bone tunnels. Composite collagen prostheses designed for ACL reconstruction degrade rapidly in bone and induce rapid ingrowth of fibrous tissue and bone. These results suggest that tissue ingrowth in the bone tunnels might provide biological fixation for collagen prostheses used for ACL reconstruction.

Optimization of extruded collagen fibers for ACL reconstruction.

Collagen fibers used in a scaffolding device for ligament reconstruction must be thin, strong, and degradable. The purpose of this study was to determine the effects of fiber diameter (20, 50, or 90 microns), crosslinking agent (uncrosslinked, dehydrothermal-cyanamide, or glutaraldehyde), and hydration on the initial mechanical properties, biocompatibility, and subcutaneous degradation rates of fibers extruded from an acidic dispersion of insoluble type I collagen. The wet tensile strength of extruded collagen fibers was significantly improved by decreasing the fiber diameter. Low-diameter, crosslinked fibers had wet tensile strengths ranging from 75-110 MPa. In contrast, high diameter fibers had wet strength values of about 30 MPa. The degradation rate of the implanted fibers, in contrast, was not significantly prolonged by changing the initial fiber diameter. This result is important because prolonged degradation of the fibers can lead to implant encapsulation instead of neoligament formation. By minimizing the diameter, fiber strength can be increased without prolonging the fiber degradation rate. Low-diameter, dehydrothermal-cyanamide crosslinked fibers have greater tensile strength and a more rapid degradation rate than medium-diameter, glutaraldehyde crosslinked fibers, and are therefore more suitable for use in a degradable ligament reconstruction device.

Mechanical and histological evaluation of amorphous calcium phosphate and poorly crystallized hydroxyapatite coatings on titanium implants.

The effect of amorphous calcium phosphate (Ca/P) and poorly crystallized (60% crystalline) hydroxyapatite (HA) coatings on bone fixation to "smooth" and "rough" (Ti-6A1-4V powder sprayed) titanium-6Al-4V (Ti) implants was investigated. Implants were evaluated histologically, mechanically, and by scanning electron microscopy (SEM) after 4 and 12 weeks of implantation in a rabbit transcortical femoral model. Histological evaluation of amorphous vs. poorly crystallized HA coatings showed significant differences in bone apposition (for rough-coated implants only) and coating resorption (for smooth- and rough-coated implants) that were increased within cortical compared to cancellous bone. The poorly crystallized HA coatings showed most degradation and least bone apposition. Mechanical evaluation, however, showed no significant differences in push-out shear strengths between the two types of coatings evaluated. Differences between 4 and 12 weeks were significant for coating resorption and push-out shear strength but not for bone apposition. Significant enhancement in interfacial shear strengths for bioceramic coated as compared to uncoated implants were seen for smooth-surfaced implants (3.5-5 times greater) but not for rough-surfaced implants at 4 and 12 weeks. Rough implants showed greater mean interfacial strengths than uncoated smooth implants at 4 and 12 weeks (seven times greater) and to coated smooth implants at 12 weeks only (two times greater). Mechanical failure of the bone/coating/implant interface consistently occurred within the bone, even in the case of the poorly crystallized HA coatings, which had almost completely resorbed on rough implants. These results suggest that once early osteointegration is achieved biodegradation of a bioactive coating should not be detrimental to the bone/coating/implant fixation.

In vitro evaluation of amorphous calcium phosphate and poorly crystallized hydroxyapatite coatings on titanium implants.

Studies of various apatite coatings on metal orthopaedic prostheses suggest that coating dissolution may promote enhanced bone bonding. Little is known concerning the effects of crystallinity and the underlying roughness on calcium phosphate (Ca/P) coating dissolution rate. To address these issues, the surface chemistry of amorphous Ca/P and poorly crystallized hydroxyapatite (HA) coatings on "smooth" and "rough" titanium (Ti) alloy (Ti-6A1-4V) implants was studied following immersion in Hank's physiologic solution at pH 7.2 and 5.2 for 0-, 4-, and 12-week periods. Changes in Calcium (Ca) ion concentrations in the solutions, coating chemistry, and surface morphology were studied by ion selective electrode, x-ray diffraction (XRD), and scanning electron microscopy (SEM) respectively. The amount of Ca dissolved from Ca/P-coated implants was strongly dependent on the chemistry of the coating and less dependent on pH or time of incubation. The effect of the underlying surface (smooth vs. rough) was not significant. The poorly crystallized HA coating underwent the most degradation, greatest crystallographic alteration, and greatest surface film formation. The amorphous coating was more stable in the saline environment, and may be more suitable in vivo if coating longevity is desired. These results suggest that this in vitro method is an effective way of determining differences in HA coating integrity.

Anterior cruciate ligament reconstruction using a composite collagenous prosthesis. A biomechanical and histologic study in rabbits.

We evaluated a prototype composite collagenous anterior cruciate ligament replacement device designed to possess the advantages of biological grafts and synthetic materials. Collagenous anterior cruciate ligament prostheses were made by embedding 225 reconstituted type I collagen fibers in a type I collagen matrix, and placing polymethylmethacrylate bone fixation plugs on the ends. The collagenous prosthesis was used to replace the anterior cruciate ligament of 31 mature rabbits. At 4 and 20 weeks postimplantation, histologic and mechanical studies were performed on the developing neoligament tissue, and compared to values for the contralateral sham-operated control. At 4 weeks, neoligament tissue infiltrated the collagen fibers of the prostheses. The tibial bone tunnel attachment site contained new bone approaching the fibrous neoligament. The glutaraldehyde-treated prosthetic fibers appeared intact, while the carbodiimide-treated prosthetic fibers began to resorb. The ultimate load and ultimate tensile strength of femur-neoligament-tibia complexes had decreased. At 20 weeks, glutaraldehyde-treated fibers appeared partially intact; in contrast, the carbodiimide-treated prostheses appeared to be completely degraded, and were replaced by organized, crimped neoligament tissue. The ultimate tensile strength and ultimate load increased substantially due to deposition and remodeling of neoligament tissue. The neoligament ultimate load was 2 to 4 times the initial load value of the prosthesis. Implantation of a resorbable, composite collagenous anterior cruciate ligament prosthesis encourages the development of functional neoligament tissue. Studies are underway to optimize the mechanical and biological properties of the prostheses.

Activities of a cancellous bone-cell-stimulating substance.

A bone-cell-stimulating substance (BCSS) that initiates appositional bone formation in intact rats was examined for its effects on DNA and collagen synthesis in tibial and calvarial organ cultures of 17-day-old embryonic chicks. BCSS stimulated collagen synthesis in both types of bone. BCSS stimulated DNA synthesis in tibiae but inhibited synthesis in calvaria from the same chicks. Insulinlike growth factor, transforming growth factor-beta, platelet-derived growth factor, and fetal bovine serum also affected DNA synthesis differently in calvaria and tibiae. BCSS was able to modify some of the effects of these growth factors on DNA.

Regeneration of Achilles tendon with a collagen tendon prosthesis. Results of a one-year implantation study.

We previously reported on the short-term biocompatibility of a reconstituted type-I collagen prosthesis that had been tested in the Achilles tendons of rabbits. Preliminary results indicated that, by ten weeks after implantation, carbodiimide-cross-linked implants had been replaced by neotendon in a manner that was similar to that of autogenous tendon grafts that had been used as controls. Also by ten weeks after implantation, glutaraldehyde-cross-linked collagen implants were encapsulated and appeared to have caused an acute inflammatory response. In the present study, carbodiimide and glutaraldehyde-cross-linked collagen implants and autogenous grafts that served as controls were implanted for fifty-two weeks as a replacement for a three-centimeter section of the Achilles tendon of rabbits. The absence of a crimp in a cross-linked implant and the presence of a crimp in normal tendon and in tendon that formed after an implant had been resorbed made it possible to distinguish between a cross-linked implant and new host tendon that had replaced the implant after it was resorbed. New collagen that had replaced the implant and autogenous (control) tendon graft were compared with normal Achilles tendon with respect to the angle and length of the crimp. The autogenous grafts and the carbodiimide-cross-linked collagen implants had been completely resorbed and replaced by neotendon. The neotendon that was present fifty-two weeks after implantation was similar, but not identical, to normal tendon. In contrast, the glutaraldehyde-cross-linked implant was essentially inert, had not been resorbed, and was surrounded by a capsule of collagenous connective tissue. The neotendon in the capsule was also similar, but not identical, to normal tendon. There were more cells in the capsule than in the autogenous grafts or in the carbodiimide-cross-linked implants. The results of the present study indicate that rapid repair is achieved with a carbodiimide-cross-linked collagenous implant that has a structure and mechanical properties that are similar to those of an autogenous tendon graft and that biodegrades at a similar rate. Prolonged biodegradation of a glutaraldehyde-cross-linked collagenous implant results in formation of a capsule and only limited formation of neotendon.

Anterior cruciate ligament replacement: a review.

The anterior cruciate ligament (ACL) is the major intra-articular mechanical element that limits motion of the tibia with respect to the femur. It is a multi-fasciculated structure composed of crimped aligned collagen fibers. The purpose of this paper is to review the literature on ACL structure and mechanical properties in an effort to stimulate the development of a new generation of more effective replacement devices. Replacement of the ACL is achieved using biologic and synthetic grafts. Biologic grafts include illiotibial band, semitendinosus and gracilis tendons, patellar tendon, and meniscus. Bone-patellar-bone complexes used to replace the ACL are revascularized and ultimately replaced by neo-ligament. Synthetic implants including the Integraft, Leads-Keio ligament, Gore-Tex¿ ligament and Kennedy Ligament Augmentation Device (LAD) have either not been approved or approved by the FDA for limited use as a replacement for the ACL. The Kennedy LAD has been found to increase the strength of autogenous tissue during revascularization. Based on the success of autografts and the Kennedy LAD, we conclude that the next generation of ACL replacement devices will consist of a scaffold and a biodegradable augmentation device. The scaffold will have a structure that mimics the normal ACL as well as stimulates revascularization and healing. A biodegradable augmentation device will be employed to mechanically reinforce the scaffold without stress shielding the neo-ligament. By combining the advantages of autografts and a biodegradable augmentation device, a new generation of ACL replacements will be achieved.

Development of a reconstituted collagen tendon prosthesis. A preliminary implantation study.

A reconstituted collagen tendon prosthesis was developed and implanted in rabbit Achilles tendons. The prosthesis was prepared by extruding type-I collagen into fibers and crosslinking it either with glutaraldehyde or with dehydrothermal treatment followed by exposure to carbodiimide. A tendon prosthesis was assembled by coating a longitudinal array of the fibers with uncrosslinked collagen. In one leg of the rabbit, the Achilles tendon was replaced with the synthetic tendon; in the contralateral leg of the animal, the tendon was excised, devascularized, and anastomosed as an autogenous graft. The autogenous tendon grafts were seen to be infiltrated centrally by fibroblasts and capillaries ten weeks postoperatively and to have been partially replaced by repair tissue twenty weeks postoperatively. Three weeks after implantation, all collagen implants were noted to have been infiltrated with fibrous tissue. At ten weeks, reorganization of collagenous tissue was observed in and around the prostheses, and the carbodiimide-crosslinked implants had been resorbed and replaced by normal-appearing neotendon. The implants that had been treated with glutaraldehyde were resorbed more slowly and were surrounded by more inflammatory cells, compared with the prostheses that had been treated with carbodiimide. Neotendon in the glutaraldehyde-treated prostheses matured more slowly. When the implants were examined at intervals after the operation, their mechanical properties approached those of fresh tendon. The initial strength of the carbodiimide-treated implants was lower than that of the fresh autogenous grafts. Twenty weeks after implantation, the strength and modulus of the carbodiimide-treated implants approached those of fresh tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

The popliteus tendon.

The posterior aspect of forty cadaver knees was dissected to determine the proximal insertion of the popliteus tendon, particularly its relationship to the lateral meniscus. Thirty-three (82.5 per cent) of the specimens demonstrated no major attachment of the popliteus tendon to the lateral meniscus. Eight specimens were from bilateral dissections and revealed no major asymmetry. On the basis of these dissections, we found no evidence that the popliteus tendon has a role in the retraction and protection of the lateral meniscus.

Bone-cell-stimulating substance.

An acid-pepsin extract of ground cancellous calf bone contains a bone-cell-stimulating substance (BCSS) that is active in the intact rat and in bone cultures. A single injection of a suspension of BCSS adjacent to the radius-ulna complex of a rat significantly increased appositional bone formation in a dose-dependent manner. The pattern of new bone deposition differed from that caused by nonspecific irritants. A suspension or water-soluble extract of BCSS significantly increased DNA and collagen synthesis in organ cultures of diaphyseal pieces of 17-day-old fetal chick tibiae. Whether or not BCSS is different from any of the other bone-derived growth factors has not been established.

Laminectomy with and without spinal fusion.

This retrospective study evaluates two groups of patients surgically treated for a symptomatic, myelographically proven, herniated nucleus pulposus with a follow-up period from 3.5 to 16 years. One hundred thirty-four patients were treated with laminectomy, discectomy, and fusion (LDF) between the years 1972 and 1978; 49 returned for follow-up examination. Two hundred patients had laminectomies and discectomies between the years 1968 and 1981; 40 returned for follow-up evaluation. Two of the disc patients had a repeat laminectomy for recurrent disc herniation, thus totaling 42 laminectomies. Each patient was scored on a scale from zero to 100. The LDF cases had an average score of 70 points. The disc cases had an average score of 69 points. Three of the LDF cases (6%) and eight of the disc cases (19%) had had a second operation. Laminectomy patients have a significantly higher reoperation rate than patients who have had spinal fusion along with laminectomy; however, the authors were unable to evaluate the results in the two groups by the overall score.

Fractures of the capitellum.

Forty intact cadaver elbows were studied to determine the contribution of the capitellum to elbow stability. With the elbow at 10 degrees of flexion, valgus motion of the elbow after capitellum excision demonstrated a minimal increase. Although some increase in valgus motion did occur after capitellum excision and radial head resection it was not until the ulnar collateral ligament was released that a severe valgus deformity was produced. In addition, isolated capitellum excisions occurring with release of the medial collateral ligament produced severe valgus motion, demonstrating the importance of medial structures to elbow stability. The cadaver study suggests excision of the capitellum in the otherwise intact elbow has little effect on valgus motion. Over the past 15 years, 17 patients with fractures of the capitellum were treated. Followup at greater than 1 year utilizing various treatment modalities is reported. Although closed reduction gave the best result, acceptable results were also obtained by open reduction and internal fixation and excision. Our clinical findings corroborated the cadaver findings in that valgus instability of the elbow only occurred when fracture of the capitellum was associated with medial ligament injuries.