Cryopreservation of intact human articular cartilage.

Damaged articular cartilage (AC) impairs joint function and many treatment techniques are being investigated to determine their long term results. Successful cryopreservation of AC can provide a reliable source of intact matrix with viable chondrocytes to maintain the cartilage over long periods of time. This study investigated the application of an established cryopreservation protocol to determine the recovery of intact chondrocytes from human AC. Ten millimeter diameter osteochondral dowels were harvested from two human donors. The cryopreservation protocol was performed and the samples were rapidly warmed from varying experimental holding temperatures (-10, -20, -30, -40 degrees C), with and without plunging into liquid nitrogen, using 1 M dimethyl sulfoxide as cryoprotectant. The cartilage was stained with membrane integrity dyes and viewed under fluorescence microscopy. The percent of intact chondrocytes was compared to fresh controls. Low recovery of intact chondrocytes was recorded from all temperature levels with and without cryoprotectant. The results of this experiment demonstrated that the cryopreservation procedure used to achieve moderate success with intact sheep AC was not successful with intact human AC and further investigation is required.

Evidence of chondrocyte repopulation in adult ovine articular cartilage following cryoinjury and long-term transplantation.

OBJECTIVE: To characterize the response of articular chondrocytes to a specific cryoinjury that leads to cluster formation following long-term transplantation. DESIGN: Osteochondral dowels from 20 adult sheep were cryopreserved to optimize the recovery of chondrocytes immediately after thawing. The dowels were transplanted as allografts and observed at 3 and 12 months. Chondrocyte distribution and viability was assessed using paravital dyes after transplantation. Chondrocyte phenotype was assessed by in situ hybridization and immunohistochemistry to detect type II collagen. An anticentrosome antibody was used to identify cells undergoing cell cycle progression towards mitosis. RESULTS: All cryopreserved grafts showed the presence of spheroidal clusters of chondrocytes 1 year after transplantation while the host cartilage adjacent to the graft appeared morphologically normal. The average size of the clusters increased from four cells at 3 months to 12 cells at 1 year. The chondrocytes in the clusters displayed newly formed type II collagen protein and mRNA. Some cells within clusters were observed with two centrosomes, indicative of cells progressing through the S phase of the cell cycle. CONCLUSION: Adult articular chondrocytes retain the ability to repopulate the matrix, an ability which is demonstrated with this specific cryoinjury. This may be an initial stage of cartilage regeneration.

An update on the nonoperative treatment of patients with metastatic bone disease.

Bone metastases constitute a major problem in oncology because of their frequency and the therapeutic problems they present. Treatment indications depend on accurate diagnosis including histologic type, number, location, and sensitivity to treatment. New findings in the pathophysiology of bone metastases, new staging procedures, new treatment modalities, and better guidelines improve therapeutic effectiveness and the quality of life of patients. There are biologic and biomechanical indications for treatment. The goals of treatment are pain relief, restoration and maintenance of function, and the prevention of complications. The nonsurgical treatment of patients with metastatic bone disease includes analgesics, hormones, radiation therapy, cytotoxic drugs, radiopharmaceuticals, chemoablation, vertebroplasty, and bisphosphonates. The future of the treatment of patients with metastatic bone disease may involve the identification of biochemical markers. The author presents an overview of the current scientific concepts of metastatic bone disease and indications and specific strategies for nonoperative treatment of patients with tumor-induced osteolysis from metastatic bone disease.

Transplantation of articular cartilage following a step-cooling cryopreservation protocol.

Using a step-cooling cryopreservation protocol that held the tissue 60 min at -4 degrees C, 30 min at -8 degrees C, and 10 min at -40 degrees C before plunging into liquid nitrogen, we were able to get a substantial improvement in the magnitude and pattern of chondrocyte recovery following cryopreservation, achieving postthaw recoveries of 62 +/- 13%. These results are consistent with the hypothesis that ice growth within articular cartilage is planar, but they provide no direct support for that hypothesis. Transplanting (step-cooled) cryopreserved osteochondral allografts into adult Suffolk/Romanoff crossbred sheep for periods of 3 months and 1 year further tested the efficacy of the cryopreservation protocol. Unfortunately, the cryoinjury sustained by the chondrocytes during cryopreservation, although apparently nonlethal immediately after thawing in many cases, was not innocuous in the long term. The presence of large clusters of chondrocytes at 1 year after transplantation illustrates that cryoinjury not detectable with a membrane integrity assay can still have far-reaching effects on transplanted tissue.

Cryobiology of articular cartilage: ice morphology and recovery of chondrocytes.

The cryopreservation of articular cartilage chondrocytes has been achieved with cells isolated from the cartilage matrix but has found only limited success when the tissue is left intact. Previous work with ovine cartilage has shown that cryopreservation of the chondrocytes of the superficial and deep zones is possible, but the cells of the intermediate zone have not been successfully cryopreserved. This finding led to the suggestion that there might be biological differences between chondrocytes of the different morphological zones that were responsible for this differential recovery. This study investigates the hypothesis that the cells of the intermediate zone are more sensitive to cryoinjury by introducing cuts in the cartilage so that cells of the intermediate zone have the same proximity to the outer surface of the tissue as the cells of the superficial zone. When this was done, it was found that cells of the intermediate zone could survive cryopreservation as well as the cells of the superficial zone when they were near a surface, but not when they were embedded deep within the tissue. Thus the hypothesis of a biological difference between the cells of the two zones being responsible for the differential recovery is disproved. It is further hypothesized that physical proximity to a surface leads to higher recovery as a result of planar ice growth into the cartilage.

Transplantation of cryopreserved osteochondral Dowel allografts for repair of focal articular defects in an ovine model.

The purpose of this study was to test whether successful cryopreservation of osteochondral tissue is possible and whether, with the appropriate surgical procedure, it can be used for the successful repair of focal articular defects within joints. Fresh (nonfrozen) and snap-frozen (plunged in liquid nitrogen and thawed in a water bath at 37 degrees C, repeated three times) autografts were used as positive and negative controls, respectively. Snap-frozen, frozen (fresh tissue placed in a freezer at -80 degrees C), and cryopreserved (immersed in 10% dimethyl sulfoxide for 30 minutes and then frozen at 1 degrees C/min to -80 degrees C) allografts were transplanted into the knees of adult sheep. Outcomes were evaluated 3, 6, and 12 months after transplantation. The morphological, histological, biochemical, and biomechanical behaviors and characteristics of the graft cartilage, the host cartilage adjacent to the grafts, and the opposing tibial cartilage were assessed. Freezing protocols that yielded poor chondrocyte recovery after thawing (frozen and snap-frozen) resulted in poor overall graft outcome. The cryopreservation protocol, however, resulted in intermediate recovery (50%) of chondrocytes and in intermediate overall graft outcome compared with fresh autografts. The membrane integrity of the allograft chondrocytes immediately following cryopreservation was identified as the most reliable predictor of long-term outcome of the graft. Further improvements in cryopreservation technique may lead to an effective method of banking osteochondral tissue for successful transplantation for the repair of focal defects and larger joint reconstructions.

Articular cartilage joint surface reconstruction techniques.

There is renewed interest in joint surface reconstruction using a variety of new and evolving techniques for articular cartilage resurfacing. Neochondrogenesis and articular cartilage transplantation are gaining a prominent place in orthopaedic basic science research. The authors have published a reliable, repeatable, stable, and sensitive model utilizing osteochondral dowel core transplantation in an ovine model to assess various treatment and follow-up evaluation techniques for articular cartilage transplantation. As well, our laboratory has developed a handheld articular cartilage indentor for clinical assessment of biomechanical performance of joint surfaces. This article presents and reviews that model as well as a semiquantitative visual analog scale for documenting articular cartilage gross morphology. The results of magnetic resonance imaging of the osteochondral dowel transplants and the gross morphology grading are compared to the histological and histochemical grading and biochemical and biomechanical assessments to form the foundation for future work in this critical and important study area for clinical application.

Osteochondral dowel transplantation for repair of focal defects in the knee: an outcome study using an ovine model.

OBJECTIVE: A model system was developed to objectively assess the quality of articular cartilage after surgical reconstruction of focal defects in the median femoral condyle using osteochondral dowel grafts. STUDY DESIGN: The surgical technique was developed and customized to reproducibly minimize surgical trauma and graft instability in order to improve the survival of the transplanted cartilage and the long-term integrity of the joint surfaces. ANIMALS OR SAMPLE POPULATION: 24 adult female Suffolk-Romanoff crossbred sheep. METHODS: Biomechanical creep testing, paravital staining for chondrocyte viability, histological analysis, and gross morphological analysis were performed at 3, 6, and 12 months postoperatively to compare fresh autografted osteochondral dowels with allografts that had been subject to a freezing protocol known to kill chondrocytes. The latter was used to investigate the time course of cartilage degeneration after injury. These two groups were also compared with normal unoperated control tissue. RESULTS: Biomechanical behavior, chondrocyte survival, and cartilage histology differed significantly between fresh grafts and those that had been frozen. CONCLUSIONS: Indentation testing and paravital staining were able to identify degenerative changes earlier than other methods of assessment. The technique developed here reproducibly and reliably transplanted osteochondral dowel grafts while minimizing the confounding effects of surgical trauma and graft instability. CLINICAL RELEVANCE: The technique provides both a promising surgical technique for the repair of focal defects of the medial femoral condyle and a sensitive model for the future study of cryopreservation strategies for articular cartilage.

Prospective cohort study of neoadjuvant treatment in conservative surgery of soft tissue sarcomas.

BACKGROUND: 1994 marked a decade since the inception of a prospective population-based study on the value of neoadjuvant approach for soft tissue sarcomas of head, neck, and limbs at the Tom Baker Cancer Centre, Calgary, Alberta. To date, 42 patients have been followed for a minimum of 5 years or until death. METHODS: Each patient received a protocol of 60 mg to 90 mg of Adriamycin infused intra-arterially or intravenously over 3 days into a vessel feeding the involved area, 30 Gy of radiotherapy given over 10 days, and complete resection of the sarcoma 4 to 6 weeks later. The lower dose was used empirically for smaller limbs (e.g., arm). RESULTS: Two of the 42 patients were immediate failures of protocol, with one requiring amputation and one requiring later reexcision. In the 38 appendicular lesions, the ultimate limb salvage rate was 97.5%. All tumors were associated with a high risk of local recurrence with 15 being previous local failures. The rest were deep and grade 2 or 3 lesions. Serious local complications were seen in one patient (2.5%) who had wound necrosis requiring reoperation. Minor wound complications were seen in five patients (12.5%) (one wound infection, one resolved edema, three long-term drainage). There was one local recurrence; thus 5-year local control was 97%. No patient had long-term morbidity related to the treatment. No effect on systemic control was suggested. CONCLUSION: Our report demonstrates that this combined modality approach provides superior local control of soft tissue sarcomas with low postoperative morbidity.

High-dose melphalan +/- total body irradiation and autologous hematopoietic stem cell rescue for adult patients with Ewing's sarcoma or peripheral neuroectodermal tumor.

The role of high-dose therapy and autologous stem cell transplantation (ASCT) in the treatment of patients with Ewing's sarcoma (EWS) remains uncertain. From November 1985 to September 1994, 13 patients aged 16-30 years (median 20.5) received high-dose melphalan (HDM) 140-200 mg/m2 +/- 500 cGy TBI followed by ASCT for relapsed/refractory (n = 4), metastatic (n = 2), or non-metastatic (n = 6) EWS, or for peripheral neuroectodermal tumor (PNET) (n = 1). This regimen was well tolerated with no transplant-related mortality and no toxicity requiring life sustaining measures. Three of the four patients treated for relapsed/refractory EWS had progression-free survivals (PFS) less than 5 months. The only long-term survivor of these four patients received HDM while in complete remission following pulmonary irradiation. Both patients with pulmonary metastases at presentation died just 5 and 6 months post-ASCT. All four patients with non-metastatic, bulky (> 8 cm) osseous EWS progressed at a median of 11 months (range 7-22 months) while the two patients with non-bulky EWS remain progression-free 25+ and 28+ months post-HDM/TBI + ASCT. The 19-year-old patient with a PNET of the thoracoabdominal wall relapsed 4 months post-ASCT. Overall, only three of these 13 patients remain progression-free at 25+, 28+, and 108+ months following HDM +/- TBI and ASCT. In conclusion, HDM +/- TBI did not obviously improve the outcome of these 13 patients relative to that expected following conventional dose therapy alone.

Limb salvage surgery for widely infiltrating bony sarcomas.

OBJECTIVE: To determine whether neoadjuvant chemotherapy and radiotherapy for bony sarcomas extending into soft tissues would allow limb salvage yet maintain local disease control. DESIGN: A prospective cohort study. SETTING: A university-affiliated cancer centre in Alberta. PATIENTS: All patients with potentially curable, widely infiltrating bony sarcomas of the extremity without neurologic deficit, referred to the centre in the 6 years from January 1984 to December 1990. There were 11 patients; 1 did not complete the protocol. The mean follow-up was 24 months. INTERVENTIONS: Adriamycin (doxorubicin) was infused for 3 days at a rate of 30 mg/d. A few days later radiotherapy was given 5 days a week for 10 doses at a rate of 3.0 Gy per dose. Four to 5 weeks later the tumour was excised surgically, with placement of a bone allograft or prosthesis, allowing a 1-cm margin of healthy soft tissue and a 5-cm margin of healthy bone and marrow cavity whenever possible. MAIN OUTCOME MEASURES: Need for limb amputation, infectious complications, recurrence of local or regional disease. RESULTS: One patient underwent amputation after fracture through the tumour site. There were two postoperative infections, one acute and one chronic. All patients had full neurologic function of the distal limb. There was no local or regional recurrence of disease. CONCLUSION: Neoadjuvant chemotherapy followed by radiotherapy and tumour excision provides control of aggressive local bone sarcomas while maintaining limb integrity.

Adamantinoma of the tibia: plain-film, computed tomography and magnetic resonance imaging appearance.

The authors describe the computed tomography (CT) and magnetic resonance imaging (MRI) characteristics of adamantinoma of the tibia in a 29-year-old man. Adamantinoma is a rare, aggressive osteolytic lesion occurring primarily in the diaphyseal portion of long bones. Because of its rarity the MRI features have been infrequently reported. In this case MRI provided more accurate information about the extent and invasiveness of the tumour than either plain radiography or CT.

Metabolic activity of bovine articular cartilage during refrigerated storage.

The ability of the chondrocytes in intact bovine articular cartilage (AC) to synthesize glycosaminoglycans (GAG) during short-term refrigerated storage was examined. Closed and exposed bovine carpometacarpal joints were stored in a refrigerator for 4 hours, 1 day, 3 days, 5 days, or 7 days after the death of the animal. Full-thickness 6 mm diameter cartilage disks were obtained from each joint, incubated in Na2(35)SO4, digested and assayed for GAG production. Similarly incubated cartilage samples were processed for autoradiography as a qualitative determination of 35S uptake by chondrocytes. All refrigerated samples of AC showed signs of some cellular metabolic activity. Only at 7 days did chondrocytes demonstrate a significant decline in activity. For all five storage periods, AC from joints exposed to nutrient media synthesized more GAG than cartilage from matched closed joints. These results suggest that some chondrocytes in AC destined for osteoarticular allografting retain the ability to synthesize GAG for as long as 5 days of refrigerated storage and that this synthesis is stimulated by storage of the joint surfaces in a sterile nutrient solution. While the implications of the chondrocytes' survival and metabolism for osteochondral allograft transplantation are unknown, these data indicate that intact bovine AC retains some metabolic activity for several days under the conditions described and would carry on this activity if transplanted within that period of time.

Ultrastructural changes of articular cartilage chondrocytes associated with freeze-thawing.

In an attempt to justify the use of cryopreserved versus fresh articular cartilage (AC) allografts, we used transmission electron microscopy (TEM) to study the ultrastructure of fresh versus frozen-thawed AC with or without a dimethyl sulfoxide (DMSO) treatment. AC explants were cut aseptically from the femoral condyles of healthy, mature rabbits when they were killed. Half of all explants were incubated in Ham's F-12 medium, supplemented with antibiotics and with or without 7.5% DMSO, frozen to -80 degrees C, stored for 24 h, and thawed rapidly. These, and the control explants, were fixed with glutaraldehyde, paraformaldehyde, and acrolein in cacodylate buffer. Sections were stained for acid phosphatase (APase), postfixed with osmium, embedded, and examined under TEM. The typical organization of the matrix and the cells was noted in control sections. The chondrocytes contained intact nuclei, organelles, and discrete plasma membrane. Although some endoplasmic reticula and nuclear membrane appeared intact, distinct ultrastructural changes were observed in frozen-thawed samples treated with DMSO. These changes included condensation of chromatin, large lipid droplets, partly disrupted plasma membrane, and pericellular precipitation of APase-positive crystalites. In sections not treated with DMSO, the cytoplasm was extensively vacuolated and no distinct organelles could be detected in the chondrocytes. Little difference was noted between the matrix organization of fresh or frozen-thawed samples. Our results suggest that distinct ultrastructural changes occur in the chondrocytes following freeze-thawing of intact AC and that DMSO pretreatment may contribute to improvement in the cryopreservation of AC.

Collagen fibril structure of normal, aging, and osteoarthritic cartilage.

The collagen architecture in normal, aging, and osteoarthritic articular cartilage was studied optically using a new silver staining technique based on specimens from 50 autopsy cases, four amputated limbs, and six osteoarthritic knees. In the normal articular cartilage, the collagen fibrils in the superficial zone were compactly arranged into layers of decussating flat ribbons mostly parallel to the artificial split lines. The fibrils showed a tendency to condense into vertical arcade columns undergirded by tangential bundles in the intermediate zone. In the deep zone, the fibrils formed a random meshwork with a slight preponderance of vertical fibrils in the perilacunar region. Three types of early degradative lesions involving the collagen network were identified. Type I lesions consisted of focal superficial disruptions related to age and friction. Type II lesions consisted of focal disruptions of tangential fibrils in the intermediate zone leading to cyst formation, probably representing a form of local stress failure. Type III lesions were found in the patella and consisted of marked swelling of the superficial zone, the cause of which was unknown. Lesions of varying severity were seen within each of the three types; the morphological changes of the more severe lesions overlapped with those of clinically overt osteoarthritis.

Neoadjuvant treatment in conservative surgery of peripheral sarcomas.

Twenty-five patients with soft-tissue and bony sarcomas of the head and neck and limbs were treated by local neoadjuvant therapy. It consisted of 90 mg of Adriamycin infused intra-arterially over 3 days into a vessel feeding the involved area and 30 Gy of radiotherapy given over 10 days; this was followed by a complete resection of the sarcoma 4 to 6 weeks later. All the tumors were associated with a high risk of local recurrence; eight were locally recurrent and the remainder were stage II to stage IV tumours. Serious local complications were seen in 4% of the patients. This rate compares well with other higher dose neoadjuvant regimens (35 Gy over 10 days), which are associated with a 35% local complication rate. Follow-up at a mean of 30 months demonstrated no local recurrence. All limbs were spared. Long-term morbidity was negligible. No effect on systemic control is suggested; only 63% of the patients were free of systemic disease. This report substantiates other similar experiences supporting neoadjuvant therapy followed by resection as the treatment of choice for local control of sarcomas.

The effect of cryopreservation on the biomechanical behavior of bovine articular cartilage.

The short-term effect of cryopreservation on specific mechanical behaviors of bovine articular cartilage has been investigated. A flat-ended nonporous indentor was used in a nondestructive, repetitive, axisymmetric unconstrained testing system. Cyclical indentation from a fixed position to a fixed load was applied until a steady-state load-deformation relationship (limit cycle) was achieved. Indentation behaviors measured from the limit cycles of each articular cartilage specimen before and after treatment were compared. Testing was done in vitro using fresh, mature bovine radiocarpal joints. Twenty pairs of cartilage-subchondral bone cores from anatomically similar sites on contralateral joints were separated into three groups; thickness controls, dimethylsulfoxide (DMSO) controls, and cryopreserved experimental samples. Thickness controls and DMSO controls were used to examine the isolated effects of the thickness measurement and DMSO incubation techniques on articular cartilage indentation characteristics. Experimental samples were cryopreserved using DMSO, their thicknesses similarly measured and indentation behaviors examined. Following testing, histological and histochemical assessment of the specimens confirmed the nondestructive nature of the tests. Intra- and intergroup comparisons of controls and experimentals revealed no statistical differences in the mechanical behaviors measured from the limit cycle or in cartilage thickness. These results indicate that the cryopreservation protocol used did not have an effect that we could measure on these specific mechanical behaviors of articular cartilage.

Investigations of low-temperature storage of articular cartilage for transplantation.

Isolated bovine articular cartilage chondrocytes and intact slices of cartilage were investigated to determine the effects of low-temperature cryopreservation on articular cartilage. Studies have focused on prefreezing conditions of cartilage, including the incubation medium and temperature of incubation, type and toxicity of the cryopreservative used, and the penetration of cryopreservative agents into cartilage cells. Cartilage freezing conditions were examined with respect to rate of freezing, controlled differential freezing rates, the ultimate storage temperature, and the time of storage. Cartilage thawing conditions were observed to ascertain the role of membrane osmotic stress during thawing and the effect of variable thawing rates on the viability of chondrocytes. Careful control of these variables can yield cartilage with cellular viability of over 50%. Optimum cryopreservation of viable cartilage should include prefreezing treatment with 7.5%-10% DMSO in nutrient medium, controlled slow freezing to -70 degrees, and rapid thawing in DMSO containing medium. A significant number of chondrocytes in deep-frozen cryopreserved articular cartilage can survive. The work recommends continued clinical use of deep-frozen cartilage.

Feline model for the study of frozen osteoarticular hemijoint transplantation: qualitative and quantitative assessment of bone healing.

Twenty-four outbred cats underwent massive osteoarticular allograft and control autograft transplantation, using the right distal femur with its articular cartilage, capsule, and medial collateral ligament intact. The cats were monitored clinically and radiographically for 1 year. Groups of cats (4 allografts and 2 control autografts) were euthanatized at 3-, 6-, 9-, and 12-month intervals. At necropsy, the grafts were photographed and assessed for bone healing and replacement by standard radiography, quantitative 99mTc bone scans, microradiography, and histologic examination of decalcified and nondecalcified specimens. The osteosynthesis site of the allografts usually healed by 5 months, compared with the autografts that healed by 3 months. As illustrated by quantitative bone scans, creeping appositional new bone slowly invaded and replaced the allograft bone. Seemingly, the cat can be used as an acceptable and clinically comparable model for the massive osteoarticular allografts currently being used for the reconstruction of joints damaged or destroyed by neoplasm surgery in limb-sparing procedures in human beings. This model may also be used to assess the rate and method of bone healing.