Luiz Augusto Ubirajara Santos

Histological study of fresh versus frozen semitendinous muscle tendon allografts

OBJECTIVE: The purpose of this study was to histologically analyze allografts from cadaveric semitendinous muscle after cryopreservation at −80°C in comparison to a control group kept at only −4°C to test the hypothesis that the histological characteristics of the tissue are maintained when the tendons are kept at lower temperatures. METHODS: In a tissue bank, 10 semitendinous tendons from 10 cadavers were frozen at −80ºC as a storage method for tissue preservation. They were kept frozen for 40 days, and then a histological study was carried out. Another 10 tendon samples were analyzed while still “fresh”. RESULTS: There was no histological difference between the fresh and frozen samples in relation to seven variables. CONCLUSIONS: Semitendinous muscle tendon allografts can be submitted to cryopreservation at −80ºC without suffering histological modifications.

COMPARATIVE STUDY OF CRYOPRESERVED BONE TISSUE AND TISSUE PRESERVED IN A 98% GLYCEROL SOLUTION

OBJECTIVE To compare the bone graft cryopreservation method (at -80 degrees C) with a preservation method using a 98% glycerol solution at room temperature (10 degrees C-35 degrees C), by testing the antibacterial and fungal effects of 98% glycerol and comparatively analyzing the observed histological changes resulting from the use of both methods. METHOD This study was of 30 samples of trabecular bone tissue from 10 patients undergoing total hip arthroplasty. Each femoral head provided 3 samples that were randomized into 3 groups, namely, the control group, the cryopreserved group, and the group preserved in a 98% glycerol at room temperature for 1 year. The samples were submitted to histomorphologic, cell feasibility, and microbiologic analyses. The results were statistically analyzed using the McNemar test, with a statistical significance index of 0.05. RESULTS Values obtained using the McNemar test to compare probability distributions of histomorphologic variables (mature or lamellar bone, immature bone, and necrosis) and cell feasibility (osteoblasts and osteoclasts) indicated that there is no difference between the distributions of variables under the 3 experimental conditions. Microbiological analysis of the 98% glycerol solution and bone fragments from samples stored for 1 year at room temperature did not show bacterial or fungal growth. The histological and microbiological investigation were performed at 2 different time points: immediately after the sample processing and after 1 year. CONCLUSION The method used to preserve bone grafts kept in 98% glycerol at room temperature (10 degrees C-35 degrees C) was similar to cryopreservation in terms of bone matrix preservation; no bacteria or fungi were found in the samples.

Development of a Fresh Osteochondral Allograft Program Outside North America

Objective To standardize and to develop a fresh osteochondral allograft protocol of procurement, processing and surgical utilization in Brazil. This study describes the steps recommended to make fresh osteochondral allografts a viable treatment option in a country without previous fresh allograft availability. Design The process involves regulatory process modification, developing and establishing procurement, and processing and surgical protocols. Results Legislation: Fresh osteochondral allografts were not feasible in Brazil until 2009 because the law prohibited preservation of fresh grafts at tissue banks. We approved an amendment that made it legal to preserve fresh grafts for 30 days from 2°C to 6°C in tissue banks. Procurement: We changed the protocol of procurement to decrease tissue contamination. All tissues were procured in an operating room. Processing: Processing of the grafts took place within 12 hours of tissue recovery. A serum-free culture media with antibiotics was developed to store the grafts. Surgeries: We have performed 8 fresh osteochondral allografts on 8 knees obtaining grafts from 5 donors. Mean preoperative International Knee Documentation Committee (IKDC) score was 31.99 ± 13.4, improving to 81.26 ± 14.7 at an average of 24 months’ follow-up. Preoperative Knee Injury and Oseoarthritis Outcome Score (KOOS) score was 46.8 ± 20.9 and rose to 85.24 ± 13.9 after 24 months. Mean preoperative Merle D’Aubigne-Postel score was 8.75 ± 2.25 rising to 16.1 ± 2.59 at 24 months’ follow-up. Conclusion To our knowledge, this is the first report of fresh osteochondral allograft transplantation in South America. We believe that this experience may be of value for physicians in countries that are trying to establish an osteochondral allograft transplant program.

Fresh osteochondral knee allografts in Brazil with a minimum two-year follow-up

Objective The present study aimed to report the results of the first series of cases of fresh ostechondral allografts in the knee joint in Brazil with a minimum follow-up of two years. Methods A protocol of procurement, harvesting, processing, and utilization of fresh osteochondral allografts in the knee joint was established, beginning with legislation modifications, graft harvesting techniques, immediate processing, storage of fresh grafts, and utilization of two surgical techniques of osteochondral transplantation. Eight patients were treated and followed-up for a minimum of two years. Results Patients were evaluated with subjective IKDC, KOOS, and modified Merle D’Aubigne and Postel questionnaires. Mean subjective IKDC score was 31.99 ± 13.4 preoperative and 81.26 ± 14.7 at the latest follow-up; preoperative KOOS score was 46.8 ± 20.9 and postoperative was 85.24 ± 13.9, indicating a significant improvement over time (p < 0.01). Mean modified Merle D’Aubigne-Postel score was 8.75 ± 2.25, preoperatively, and 16.1 ± 2.59 postoperatively. Friedman test for non-parametric samples demonstrated a significant improvement in postoperative scores (p < 0.01). Conclusion The use of fresh osteochondral allografts in Brazil is a safe procedure, with good clinical results in the short- and medium-term for the treatment of osteochondral lesions greater than 4 cm2 in the knee joint.

Cryopreservation - A Viable Alternative in Preparation for Use of Allografts in Knee Ligament Reconstruction

The first report of the use of allografts in humans dates back to 1881. The first tissue bank of bone grafts was created in 1940 in the United States and the initial clinical results were published in 1942 by Inclan, 1942. Since then a series of regulations and studies has emerged relating to the use of grafts in orthopedic practice19. Currently, tendon allografts are used in knee surgeries, in elbow ligament reconstructions and in revisions of the acromioclavicular joint (Costic et al., 2004).

Cryopreserved Musculoskeletal Tissue Bank in Dentistry: State of the Art and Perspectives

Maxillary and mandibular bone loss has long been a challenge to dental surgeons who seek to reconstruct these lost segments. These lesions lead to deformation of some maxillary and mandibular areas which interferes in the functional rehabilitation process of these structures. The most common cause of these lesions is prolonged use of total prostheses in a large part of the Brazilian population and the searches for surgical techniques and bone substitutes are today proposed and studied by the academic class. In this context, Brazil is starting to distribute allogeneic tissue obtained, processed and qualified by musculoskeletal tissue banks. Such banks already have experience in dispensing tissue to the orthopedic area, which has been using reconstructive techniques with allografts for many years. The first studies proposing the use of bone substitutes for replacement of these faulty parts commenced in the decades subsequent to 1860. (Carrel, 1912;Groves, 1917; Sharrard, Collins, 1961; Urist, 1965; Fischer, 1998; Tomford, 2000).

Validation of Primary Packaging for Cryopreserved Musculoskeletal Tissues

Bones and tendons are obtained from donors who have been pronounced brain dead after a rigid and extensive screening process. The tissues obtained are sent to a Tissue Bank and submitted to processing steps, packaging and cryopreservation at -80◦C. It is vital to maintain sterility and integrity, so that no tissue is discarded.These precautions also extend to the packaging, which should promote containment and protection. Although minimum technical criteria have already been defined for the food industry, this has still not been regularized in Brazil. We emphasize, therefore, the need to study this subject, focusing on maintaining the quality of the musculoskeletal tissues produced by tissue banks. All procedures developed byTissue Banks currently present in the country have rigid control over the quality and the traceability of tissues made available, based on international standards (EATB, 2004; AATB, 2007) on the legislation (BRASIL, 1997-2006) and in conformity with Good Manufacturing Practices GMP.