Objective: To define histological scores for intervertebral disc degeneration that would enable the definition of morphological characteristics of disease, besides improving knowledge of the lumbar degenerative disc disease by means of immunohistochemical markers. Methods: Hematoxylin and Eosin, Alcian/PAS, Masson Trichrome and Safranin O/FCF staining was used on the intervertebral disc degeneration sections of patients with lumbar degenerative disc disease. The protein markers defined in immunohistochemistry were cell proliferation (Ki-67) and apoptosis (p53). Results: The study data enabled the determination of Safranin O/FCF stain as the most effective one for evaluating parameters such as area, diameter, and number of chondrocyte clusters. The importance of using stains in association, such as Safranin O/FCF, Masson Trichrome, Alcian/PAS and Hematoxylin and Eosin, was also determined, as they are complementary for the histopathological verification of intervertebral disc degeneration. By expressing proteins using the immunohistochemistry technique, it was possible to consider two stages of disc degeneration: cell proliferation with chondrocyte cluster formation, and induction of apoptosis. Conclusion: This study enabled the histological and immunohistochemical characterization to be determined for lumbar degenerative disc disease, and its degrees of evolution, by determining new disc degeneration scores.Keywords: Intervertebral disc; Histology; Immunohistochemistry; Cell proliferation; Apoptosis. RESUMO

To review the potential role of stem cells in treating degenerative disc disease of the intervertebral disc (IVD). A review was performed of articles from the Medline database concerning stem cells and degenerative disc disease (DDD). To discuss the data, the papers were classified as: review, in vitro, experimental, and clinical. The currently available treatments were basically for symptom reduction, not to revert the IVD degenerative process. The use of mesenchymal stem cells (MSC) is being proposed as an option of treatment for DDD. In vitro studies have shown that the MSC are able to differentiate into NP cells and that the MSC also reduce the inflammatory levels of the degenerated IVD. Besides, experimental studies demonstrated that the MSC remained viable when injected into the IVD, and that they were able to regenerate partially from the degenerated IVD and its structure. The few clinical studies found in the literature presented diverging results. The use of MSC is being widely studied and shows promising results for the treatment of DDD. Although many advances are being achieved in studies in vitro and experimental, there is a lack of clinical studies to prove the role of MSC in DDD management.

Objectives This study aimed to evaluate the efficacy of hyaluronic acid in the viability and proliferation profile of human femoral-tibial joint cartilage affected by osteoarthritis using in vitro models of chondrocytes in a 2-dimensional (2D)- and 3-dimensional (3D)-based culture model by spheroids. Design In vitro study of knee cartilage affected by osteoarthritis that required surgical treatment. Samples were cultured and exposed to hyaluronic acid (100 and 500 μM; intervention group) or vehicle solution. In monolayer or 2D culture, proliferation and cell viability were measured, and nuclear morphometry was analyzed by 4′,6′-diamino-2-fenil-indol (DAPI) staining. The 3D-based culture established from the culture of articular cartilage of patients submitted to total knee arthroplasty evaluated the diameter, viability, and fusion ability of the chondrospheres created. Results Samples from 3 patients resulted in viable cultures, with chondrocyte cells exhibiting a potential for cell proliferation and viability to establish a culture. Hyaluronic acid (100 and 500 μM) improved chondrocyte viability and proliferation up to 72 hours in contact when compared with the control group, and no nuclear irregularities in morphology cell characteristics were observed by DAPI. In the 3D evaluation, hyaluronic acid (500 μM) improved the cellular feedback mechanisms, increasing the survival and maintenance of the chondrospheres after 7 days of analysis, showing the intrinsic capacity of chondrospheres grouped in the attempt to rearrange and reestablish new articular tissue. Conclusions The 2D- and 3D-based culture models with hyaluronic acid improved chondrocyte viability and proliferation and demonstrated the ability of freshly formed chondrospheres to undergo fusion when placed together in the presence of hyaluronic acid.

Objective: To evaluate the locomotor and histological impact on the spinal cord comparing lateral and posterior clip placement. Method: Randomized experimental trial. Twenty female Wistar rats, weighing between 250 and 300 grams and aged 12-14 weeks were randomized in two groups according to the placement of the clip: lateral group (N=10) and posterior group (N=10). After exposing the thoracic segment of the spine (T8-T10), a laminectomy was performed at the T9 level under microscopic view. The spinal cord injury was made using a 5 mm long aneurysm clip with a closing pressure of 50 grams. Locomotor behavior was evaluated by the Basso, Beattie and Bresnahan scale in days 1, 7, 14, 21, and 28 after surgery. The area of injury was assessed by histological analysis and measured by a software. Results: The histological evaluation showed a larger mean area of 4.8±1mm² of lesion (P=0.03) in the lateral group when compared with the posterior group mean area of 2.3±2mm². There was no significant difference between lateral and posterior groups with respect to locomotor scores from day 1 to 28 (P=0.361). Conclusion: The lesion area observed in the spinal cord histology after lateral placement of a clip was significantly bigger than in the posterior placement. The motor evaluation showed similar BBB scores regardless of the type of clamping method.

Introduction Literature on histological and immunohistochemical protocols that relate degenerative discopathy and its degree of wear is scarce. The purpose of this study was to analyze the structural degeneration of the intervertebral disc of patients with different degrees of degeneration, through histopathology, immunohistochemical, and cellular proliferation and apoptosis studies. Materials and Methods The intervertebral disc was obtained from patients with lumbar disc degenerative disease refractory to conservative treatment who required surgical treatment. The material was sent for histological study with hematoxylin and eosin stain (H&E), Alcian/PAS, Masson trichrome, and safranin O/FCF, and immunohistochemistry through expression of the proteins to mark cell proliferation, Ki-67, and apoptosis, p53. MRI defined the degree of disc degeneration according to Pfirmann classification, and it was correlated with the evaluations of the different histological stains, and the positivity of the immunohistochemical markers. The present study was authorized by the ethics committee under number 0513. Results H&E was effective to evaluate fissures of the fibrous ring, necrosis, and inflammation. Alcian/PAS stain allowed a better visualization of the chondrocyte clusters and of the acid mucopolysaccharide deposits in 86.7% of the samples. Masson trichrome identified the misalignment of collagen fibers in 93.4% of the samples, with greater definition. The safranin technique was considered the most sensitive and efficient stain to study the number and diameter of chondrocyte clusters. Immunohistochemistry showed 73% of positive expression of Ki-67 and p53, which are related, respectively, to the cell proliferation followed by cellular apoptosis. Conclusion The protocol enabled a better histological and molecular characterization of the degenerative disc according to different stages of degenerative disc disease. Disclosure of Interest A. Falavigna: Conflict with CNPq, FAPERGS, and AOSpine Latin America M. Peletti-Figueiró: None declared I. S. Aguiar: None declared M. Roesch-Ely: None declared D. C. Machado: None declared J. A. P. Henriques: None declared O. Righesso: None declared