Culture of human bone marrow-derived mesenchymal stem cells on of poly(L-lactic acid) scaffolds: potential application for the tissue engineering of cartilage.

PURPOSE: Due to the attractive properties of poly(L-lactic acid) (PLLA) for tissue engineering, the aim was to determine the growth and differentiation capacity of mesenchymal stromal cells (MSCs) in PLLA scaffolds and their potential use in the treatment of cartilage diseases. METHODS: MSCs were cultured in PLLA films and thin porous membranes to study adherence and proliferation. Permeability and porosity were determined for the different scaffolds employed. The optimal conditions for cell seeding were first determined, as well as cell density and distribution inside the PLLA. Scaffolds were then maintained in expansion or chondrogenic differentiation media for 21 days. Apoptosis, proliferation and chondrogenic differentiation was assessed after 21 days in culture by immunohistochemistry. Mechanical characteristics of scaffolds were determined before and after cell seeding. RESULTS: MSCs uniformly adhered to PLLA films as well as to porous membranes. Proliferation was detected only in monolayers of pure PLLA, but was no longer detected after 10 days. Mechanical characterization of PLLA scaffolds showed differences in the apparent compression elastic modulus for the two sizes used. After determining high efficiencies of seeding, the production of extracellular matrix (ECM) was determined and contained aggrecan and collagens type I and X. ECM produced by the cells induced a twofold increase in the apparent elastic modulus of the composite. CONCLUSIONS: Biocompatible PLLA scaffolds have been developed that can be efficiently loaded with MSCs. The scaffold supports chondrogenic differentiation and ECM deposition that improves the mechanics of the scaffold. Although this improvement does not met the expectations of a hyaline-like cartilage ECM, in part due to the lack of a mechanical stimulation, their potential use in the treatment of cartilage pathologies encourages to improve the mechanical component.

Scanty integration of osteochondral allografts cryopreserved at low temperatures with dimethyl sulfoxide.

PURPOSE: To compare the integration of osteochondral allografts cryopreserved at different temperatures and different concentrations of dimethyl sulfoxide in an in vivo sheep animal model. METHODS: Thirty-six adult sheep were randomly allocated to 6 groups of allograft osteochondral transplantation. Six osteochondral cylinders were stored for 6 weeks at -80°C; 6 at -80°C with 10% dimethyl sulfoxide (DMSO); 6 at -80°C with 10% DMSO for 90 min; 6 at -186°C; 6 at -186°C with 10% DMSO; 6 at -186°C for 90 min. After transplantation, all animals were euthanized at 6 months. Harvested specimens underwent gross morphologic and histologic evaluation. RESULTS: We found no statistically significant differences when comparing the gross cartilage morphology and histopathologic scores of each group. The Mankin and OARSI scores and the modified Wakitani and OARSI scores showed a good correlation grade. The Mankin and modified Wakitani scores showed a fair correlation grade. CONCLUSION: The cryopreservation protocols adopted in the present study provided scanty integration in an in vivo sheep model of osteochondral allograft transplantation. Therefore, their use in the clinical practice is discouraged.

Gene expression and proliferation analysis in young, aged, and osteoarthritic sheep chondrocytes effect of growth factor treatment.

Cartilage is a support tissue with a poor capacity to self-repair. Its cells, chondrocytes, are responsible for synthesizing and renewing the matrix that surrounds them in a constant turnover mechanism. Autologous chondrocyte implantation (ACI) is one of the techniques that promises to be an alternative to common strategies for chondral lesions. To apply this technique, a large amount of cells must be obtained. In our work, we studied the state of cells from different cartilage (young, aged, and osteoarthritic sheep) cultured in monolayer by analyzing their proliferation rate using bromodeoxyuridine and their gene expression profile by RT-PCR. A decrease was found in expression of type II collagen and aggrecan in aged, osteoarthritic, and passaged chondrocytes. Treatment of cells with growth factors aFGF, IGF-I, TGF-beta, and OP-1 improved the proliferation rate in all cells studied and stimulated gene expression of type II collagen, aggrecan, and TGF-beta. Osteoarthritic cells showed a poor response according to matrix gene expression, while young cells responded properly, and aged chondrocytes showed a moderate response. These results suggest that the state of cartilage may affect the behavior of cultured chondrocytes.

Articular cartilage degeneration after frozen meniscus and Achilles tendon allograft transplantation: experimental study in sheep.

PURPOSE: The purpose of this study was to analyze cartilage degeneration in knees after total medial meniscectomy, transplantation of fresh-frozen meniscus allograft, and Achilles tendon allograft. TYPE OF STUDY: Experimental study. METHODS: We have studied the articular cartilage in the medial compartment of the left knees in 32 sheep aged 5 to 6 months, with 8 animals in each group. The study was performed after meniscectomy (group I), transplantation of fresh-frozen meniscus allograft (group II), use of fresh-frozen Achilles tendon allograft (group III), and in a control group (group IV). For the histologic study, all samples were stained with Masson's trichrome and Safranine-O. Mankin's score was applied to grade the histologic damage to the articular cartilage. RESULTS: The group with the greatest number of degenerative changes was group III, followed by groups I and II. The percentage of thickness of cartilage detected by Safranine-O stain was found to be significantly different in both tibia and femur between the control group and the other 3 groups, but not among groups I, II, and III. The immunoreactivity of the articular surfaces in tibia and femur showed notable differences in all the groups. Collagen X was present in the degenerative hypertrophic chondrocytes in the damaged articular surfaces. CONCLUSIONS: Meniscal replacement with meniscal and Achilles tendon allografts provides partial protection against articular damage after a meniscectomy.

Meniscal repair possibilities using bone morphogenetic protein-7.

This study analysed the influence of bone morphogenetic protein-7 (BMP-7) on cells and meniscal structure. The effect of treatment with BMP-7 was assessed in vitro and in vivo in lesions in the avascular area of the meniscus. Cells were extracted from the outer and inner part of eight menisci of four 2-year-old merino sheep. The menisci were digested with a collagenase mix, and meniscus cells of the synovium, vascular area and avascular area were extracted. The expression of genes for collagen (Col1 and Col2A), matrix metalloproteinases (MMP-2 and MMP-13) and aggrecan was analysed by real time quantitative polymerase chain reaction (qPCR) at baseline and after incubation with BMP-7. Eight sheep aged 2 years and weighing 35-40 kg were used for the in vivo study. Surgery was performed in both knees of every animal. Two holes were made in the avascular area of the medial meniscus of both knees and filled using Putty(®) (control groups) or OP-1 Putty(®), which comprises BMP-7 mixed with a cellulose putty carrier (experimental groups). Animals were sacrificed at 6, 12 and 25 weeks. Adding BMP-7 to vascular cells of the meniscus was associated with a 15-fold increase in Col2A expression and a 78-fold increase in BMP-7 expression. BMP-7 inhibited MMP-2 and MMP-13 expression. Adding BMP-7 to synovial cells inhibited the expression of Col1, doubled the expression of Col2A and reduced the expression of BMP-7; the expression of MMP-2 was inhibited, while that of MMP-13 was increased three-fold. Incubation of cells from the avascular region with BMP-7 was associated with a 2.4-fold increase in Col1 expression, and a 4.4-fold increase in Col2A expression compared with the control. The expression of MMP-2 and BMP-7 was inhibited. In the in vivo study, treatment of the holes in the avascular area of the meniscus with BMP-7 was associated with an important cell presence inside the holes and the appearance of fibrous tissue after 12 weeks; these features were not seen in the control groups. BMP-7 may be a suitable growth factor for stimulation of meniscal cell and collagen formation.

Platelet-rich plasma, rhOP-1 (rhBMP-7) and frozen rib allograft for the reconstruction of bony mandibular defects in sheep. A pilot experimental study.

A 6 cm bony defect in the mandible of 15 sheep, 8 years old, was reconstructed using variously allograft of frozen rib, rhOP-1 (rh BMP-7), platelet-rich plasma (PRP), and a combination of frozen rib allograft and rhOP-1. The histological, histomorphometric, immunohistochemical and radiographic features of reconstruction were analysed. The animals were euthanised at 2 months postoperatively. In the control and PRP groups, no bone formation was detected. The sheep receiving rhOP-1 showed some and those receiving both rhOP-1 and allograft showed most new bone formation; in both groups this was through endochondral and also fibrous ossification. The combination of bone allograft with growth factors demonstrated osteoconductive as well as osteoinductive properties, and is appealing in the management of problem fractures.

In vitro healing of avascular meniscal injuries with fresh and frozen plugs treated with TGF-beta1 and IGF-1 in sheep.

We studied the effect of freezing and inserting meniscal plugs in lesions generated in the avascular area of sheep menisci maintained in vitro, and whether the healing process can be improved by adding growth factors TGF-beta1 and IGF-1. Thirty six menisci obtained from healthy 3 months-old sheep were cultured in 6 well plates and holes were perforated in the avascular area. Meniscal plugs, either fresh or frozen at -20 degrees C for 1 month, were used to fill in the lesions, and then cultured in the presence or absence of TGF-beta1 or IGF-1 for 8 weeks. Samples stained with Massons trichrome were analyzed to evaluate the attachment of the plug and the cell density of the tissue. BrdU immunohistochemistry was performed to identify the proliferation of meniscal cells. Both growth factors improved considerably the cell density of implanted plugs. TGF-beta1 increased significantly the attachment of both fresh and frozen plugs, but it had no effect on meniscal cell proliferation. In contrast, IGF-1 had no effect on the attachment, but did increase significantly the number of proliferating cells in the surface of the host meniscus and the inserted plug. In conclusion, frozen plugs can survive if treated with either TGF-beta1 or IGF-1. The combination of TGF-beta1 and IGF-1 could aid in the repairing of the avascular meniscal injuries, as they are capable of promoting the attachment of tissue, and increasing the proliferation of meniscal cells.

IGF-1 gene therapy to protect articular cartilage in a rat model of joint damage.

INTRODUCTION: An adeno-associated virus (AAV) derived vector in gene transfer model that induces IGF-1 expression could repair articular cartilage. MATERIALS AND METHODS: Male Wistar rats, 150 and 200 g, and 7 weeks old, were used. Effectiveness of constructed vectors was assayed inoculating them in rat knees of control and damaged animals either mechanically or by collagen-induced arthritis. Inoculation was intra-articular with 50 microL of recombinant AAV-Luciferase (1.25 x 10(8) particles). The rats were killed after 1, 2, 4 and 8 weeks. IGF-I activity was analyzed by injecting 50 microL of recombinant AAV (1.25 x 10(8) particles) in animals with damaged knees. Final analysis was performed after 8 weeks. RESULTS: The activity of AAV vectors in vitro shows the presence of mRNA coding to IGF-I in cells infected with AAV-IGF and not in control cells without viral vectors and an increase in secreted IGF-I protein in culture medium. In vivo, AAV derived vectors induced protein expression in cartilage 2 months after inoculation. In the animals killed after 1 and 2 weeks, no significant increase in the reaction of luciferase was observed (P > 0.05). In the group of animals with no injury an increase was observed at 4 weeks, which was more marked and significant after 8 weeks (P = 0.029). The same behavior occurred in the animals with induced arthritis and in the mechanical injury group. In the levels of expression after 8 weeks, no significant differences were found between the two groups of injured animals and the group of healthy animals infected with the virus. The joints of the animals that were subjected to injuries in the cartilage and inoculated with AAV-IGF-I presented a similar appearance to those animals inoculated with saline solution. CONCLUSION: Autoimmune and mechanical lesions did not show improvement in the state of its cartilage after the treatment. The use of AAV vectors capable of inducing the expression of IGF-I in vitro is therefore not sufficient to protect the cartilage from the serious damage.

Cell viability and protein composition in cryopreserved cartilage.

Chondrocyte survival in frozen-stored osteochondral allografts is low. We analyzed different storage conditions to determine the best for maintenance or storage for preserving cartilage. We hypothesized cell viability and extracellular protein content could be improved with better cryopreservation. Articular cartilage from nine sheep femoral condyles were stored at 4 degrees C, -80 degrees C, and -196 degrees C with and without cryopreservative agents for 1 month. We determined cell viability and performed Western blot analysis for TGF-beta, IGF-1, and MMP-2, 9, and 13. We observed decreases in viability in cartilage stored at 4 degrees C: 36.2% viability when stored without solution, 40.4% in phosphate-buffered saline, and 48.1% in culture medium. TGF-beta and IGF-1 decreased in the first week in all groups. The groups stored at -80 degrees C and -196 degrees C contained less protease in the last 2 weeks. In the groups stored at 4 degrees C in phosphate-buffered saline, we detected no increase in the levels of MMPs. Our data discourage the use of frozen cartilage because of the decrease of cell viability and elevation in MMPs. However, modifying the freezing conditions might moderate these changes and improve the state of preserved cartilage.

Morphologic comparison of cervical, thoracic, lumbar intervertebral discs of cynomolgus monkey (Macaca fascicularis).

The aim was to analyze the morphological differences of the intervertebral disc and endplates at different levels. Forty-five vertebral motion segments were obtained from the spine of nine 3 to 4-year-old cynomolgus monkeys (Macaca fascicularis). From every spine, five discs were sectioned (C5-C6, T3-T4, T9-T10, L2-L3, L4-L5). In all the groups, tissue samples were collected and sections were stained with Masson's trichrome, Safranine-O and van Gieson's connective tissue stain to analyze the intervertebral discs. Immunohistochemistry was performed, using specific antibodies to detect collagens I and II. The intervertebral disc height, the maximum nucleus pulposus height, the superior and inferior endplate heights were histomorphometrically measured and different indexes were calculated to compare the differences between specimens of the same animal and between discs of the same level, and finally the differences between groups of discs of different levels. There were no differences existing in annular fibers anchoring on the endplate between discs of different levels. A positive immune reaction for type I collagen was observed in the longitudinal ligaments and in the annular region adjacent to them. Collagen II immune reactivity was found in the annulus close to the nucleus pulposus, in the endplates and in the nucleus. There were no differences between discs of different levels in the collagen I and II localization. The height of the discs varied along the spine. The smallest value was measured in T3-T4, with a larger increase caudally than cranially. The highest value was measured in L2-L3. A cervical disc was 55% the height of a lumbar one. The endplate height increased along the length of the spine. The inferior EP was always higher than the superior. The study provides a detailed structural characterization of the intervertebral disc and may be useful for further investigations on the disc degeneration process.

Radiological and histological analysis of cortical allografts: an experimental study in sheep femora.

INTRODUCTION: We developed an experimental model in sheep femora to evaluate the process of cortical allograft incorporation. MATERIALS AND METHODS: Twenty-four sheep were divided into four groups according to the various treatments of cortical allografts as follows: fresh, frozen, autoclaved, and frozen with perforation. Periodical radiographic and histological evaluations were performed for each group. RESULTS: Perforated frozen allograft proved to be superior radiographically in the first stage to fresh, frozen, and autoclaved forms. Revascularization was demonstrated by both Spalteholz's technique and histological examination. Histological analysis also showed creeping substitution, from the host bone to the allograft, which increased the reabsorption to facilitate new bone penetration, including endochondral ossification at the host-graft interface. CONCLUSION: We believe that endochondral ossification is probably a biological event occurring routinely during the bone healing process and that the processes of incorporation of variously treated cortical allografts differ only at the early phase of implantation.

Efecto del almacenamiento de menisco a 4ºC sobre la morfología y la capacidad de respuesta celular a los factores TGF-ß1, IGF-1, aFGF, bFGF y BMP-7 en cultivo en monocapa / Effect of meniscus storage at 4ºC upon cell morphology and capacity to respond to growth factors TGF-ß1, IGF-1, aFGF, bFGF and BMP-7 in monolayer culture

Objetivo del trabajo: Estudiar la viabilidad del almacenamiento de menisco a 4ºC. Material y método: Se obtuvieron fragmentos de menisco procedentes de corderos sanos en condiciones de esterilidad. Como control, se utilizaron fragmentos de tejido digeridos inmediatamente después de la extracción del menisco, el resto se almacenaron a 4ºC sumergidos en medio de cultivo durante 15 y 30 días. Tras el almacenamiento, se realizó la digestión del material utilizando tripsina, colagenasa y dispasa y se cultivaron las células en monocapa. Se estudió la morfología celular al microscopio óptico y la respuesta a los factores de crecimiento TGF-ß1, IGF-1, aFGF, bFGF y BMP-7 mediante ensayos de proliferación. Resultados: La respuesta celular a bFGF e IGF-1 se incrementó tras los 15 días de tratamiento, para volver a descender tras los 30 días. La capacidad de respuesta tras el tratamiento con BMP-7 descendió a los 15 días y volvió a ser similar al control a los 30 días. Los factores aFGF y TGF-ß no modificaron la respuesta que se produjo en las células. Conclusiones: Tras el almacenamiento a 4ºC las células sufren cambios morfológicos y bioquímicos que las lleva a modificar su sensibilidad a algunos factores de crecimiento (AU)

Aim: To evaluate the viability of meniscus storage at 4ºC. Material and method: Meniscus fragments were obtained from healthy goats under sterile conditions. As control, use was made of tissue fragments digested immediately after meniscus harvesting; the rest were stored immersed at 4ºC in culture medium for 15 and 30 days. Following storage, the material was digested using trypsin, collagenase and dispase, with monolayer culture of the cells. Cell morphology was evaluated under the light microscope, and cell response to growth factors TGF-ß1, IGF-1, aFGF, bFGF and BMP-7 was assessed by proliferation tests. Results: The cell response to bFGF and IGF-1 increased after 15 days of treatment, followed by a decrease after 30 days.The response capacity after BMP-7 treatment decreased after 15 days and returned to control levels after 30 days. Factors aFGF and TGF-ß did not modify cell response. Conclusions: Following storage at 4ºC, the cells undergo morphological and biochemical changes that modify their sensitivity to certain growth factors (AU)