DECRETO 0724 DE 2012 PDF

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Osteoarthritis OA is a degenerative joint disease with no efficient treatment due to limited endogenous regenerative capacity. It affects the knees of nearly a quarter of the population aged 60 and older [ 12 ].

Currently, patients with severe OA are inexorably relegated to prosthetic joint substitution.

OA is a disorder involving movable joints characterized by cell stress and extracellular matrix degradation initiated by micro- and macroinjury that activates maladaptive repair responses including proinflammatory pathways of innate immunity.

Since the OA pathological processes are well described involving among others, the loss of functional chondrocytes, several cell-based therapeutic approaches have already been successfully developed including bone-marrow stimulation [ 5 ], implantation of osteochondral autograft [ 6 ] or allografts ACI [ 7 ], and transplantation of expanded autologous chondrocytes [ 8 ], or amplified mesenchymal stem cells MSCs [ 9 ], which help to restore articular cartilage.

The intra-articular administration of MSC directly in the synovial fluid has been the predominant cell-based approach, with already demonstrated clinical effectiveness, with probed regenerative and immunosuppressant activities [ 910 ]. To date, it continues to be an important avenue fe research and clinical development 201 to its extraordinary therapeutic aptitude. However, the direct differentiation of multipotent MSC into cells of the chondrogenic lineage decreho led to a variety of experimental strategies to investigate whether tissue-specific MSCs are preferential for the regeneration and maintenance of articular cartilage [ 9 ].

Additional efforts on engineered cartilage implants are also done by using MSC synergistically activated with biomolecules to potentially improve chondral and osteochondral lesion repair, converting those in specialized trophic producers to initiate endogenous regenerative activities in the OA joint [ 11 — 13 ]. The long-term durability, increased tissue integration, and specific activity depend first on better transplantation survival rates and better adaptation to the hostile environment which still comprises a challenge.

To date, there is not a precise description of which donor sample would be more efficient in the generation of MSC for the treatment and repair of joints in a degenerative process. Subcutaneous fat tissue is the most accessible source; however, after prosthetic implementation for joint substitution, the supra- and infrapatellar fat pads are commonly resected, constituting a suitable autologous adipose-derived MSC source ASC.

The suprapatellar or quadricep fat pad is externally interposed between the joint capsule and the synovium, lined to the joint cavity showing a triangular shape and extended through the patellar base [ 15 ].

Previous studies have already described the potential regenerative capability of ASC derived from the infrapatellar pad in a model of OA [ 1617 ], showing in fact a higher percentage of immunophenotypical positive stromal cells in comparison with those obtained from subcutaneous fat [ 18 ]. However, no previous data has been reported regarding the suprapatellar tissue regenerative capacity.

There are stem cell niches with no spontaneous capacity to mobilize to decretl injury to repair the 201 OA defects. The vecreto of these cells from these specialized sources could render an optimal source of dd or even allogenic applications to promote cartilage regeneration. Indeed, the patellar fat pad-derived ASC would offer a potential autologous regenerative treatment for patients with multiple degenerative OA. Twenty-four patients between 50 and 80 years old indicated for complete joint substitution were included in the study.

The samples, adipose tissue from supra- or infrapatellar areas, were anonymized and individually housed and collected inside the surgery room decrero sterile containers with sterile saline.

As exclusion criteria, no samples were collected from patients with a history of cancer and infectious diseases active at the time of the surgery viral or bacterial.

The samples were washed multiple times in PBS plus antibiotics to clean the tissue and remove residual blood. The following day, the digested adipose tissue was collected decreti washed multiple decreeto with PBS plus antibiotic by serial centrifugation. For FACS analysis, 10 5 cells were utilized for each pair of antibodies. The following day, the medium was removed and replaced with fresh medium and attached cells were allowed to grow until nearly confluent and then subjected to cell proliferative analysis, clonicity assay, morphological assessments, and FACS analysis and cell differentiation assays.

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As a negative control, cell suspension without antibody was employed following the same procedure. The amplification 072 expansion of the ASC population from the SVF involves first the colony forming units decrero attachment onto a substrate and then a subsequent amplification in the presence of appropriate growth factors [ 23 ].

The medium was replaced every third day. The excess fe was removed by subsequent washes with tap water. The cells were allowed to air dry and then visualized under the microscope. Cells were seeded for up to ten days of analysis, by quantifying the number of cells in a Neubauer chamber every day in every well.

Finally, cells were rinsed in distilled water, dehydrated in ethanol, and infiltrated overnight in Durcupan resin Fluka, Sigma-Aldrich, St. Following polymerization, embedded cultures were detached from the chamber slide and glued to dw blocks.

Serial semithin sections 1. ASC at or after passage 4 were subjected to directed differentiation [ 24 ], to induce adipogenesis, osteogenesis, and chondrogenesis, for each sample, supra- and infrapatellar from three different patients. All directed-differentiation media were obtained from Lonza Lonza Co.

Decreto 0724 de 2012 movie

The cells were then incubated in standard cell culture conditions for 12 days. Serial washes with water to remove the excess staining were applied.

ASC cultures were maintained in this medium for 4 weeks with medium changes every 3 days. Se detection of extracellular calcium deposits, Alizarin red staining was used in formalin-fixed cultures; Alizarin red solution 0.

Excess dye was removed by several washing steps using water. The orange-red calcium precipitates were dw from at least four different pictures of each sample by using ImageJ software. Blue staining was quantified from 4 different pictures of each sample with ImageJ software.

The PRP was isolated from blood collected from all patients decreho citrate tubes and was prepared following the standardized method described in Anitua et al. All animals were sacrificed one month after OA induction, and knee joints were collected.

The leg was isolated and osteomized in the middle of the femoral shaft and in the middle of the tibia and fibula to get the whole joint. Most of the adherent connective tissue, including muscle, ligaments, and tendons, surrounding the knee were removed preventing any damage to the cartilage. One knee was used for histological analysis decreeto the other knee for the quantification of glycosaminoglycans GAGs. Immediately after tissue dissections, GAGs were individually quantified for tibia and femur portions from one treated knee of all animals in all groups.

Weighted samples were digested in 2. A chondroitin sulfate standard curve was prepared in parallel. Dehydrated samples were mounted in Eukit. Bright-field pictures of all series every 4th section were acquired for OA score quantification following the recommendations established by Glasson et al. The entire joint was analysed at all four quadrants and through multiple step sections through the joint following the 0—6 subjective scoring system, where 0 is normal; 0.

Both fixed cells on a cover slip from osteogenic-directed differentiation or the chondrogenic-directed differentiated as well as fixed bone after demineralization by EDTA immersion paraffin-embedding sections were subjected to protein expression analysis by specific immunostaining.

The paraffin-embedded sections were first deparaffinized. Tissues were incubated with Oregon Green dye conjugated goat anti-rabbit IgG 1: Signals were visualized by confocal microscopy Leica, Germany ; at least 6 different fields per condition and assay were analysed.

1. Introduction

The deecreto was i. In all cases, the whole body PET scan time was 10 minutes. The supra- and infrapatellar fat pads of twenty-four patients with severe OA were resected during the surgical intervention for prosthetic implantation.

After tissue processing, total cell counting rendered no significant differences in the amount of nucleated cells between the supra- or infrapatellar samples 7. No significant differences were detected in any other assayed marker, including the hematopoietic precursor cell marker CD34 or the mature leucocyte cell marker CD Supra- and infrapatellar fat pad cell populations in the SVF.

The positive cell populations were expressed as a percentage of the total analysed cell population. ASC cultures derived from both supra- and infrapatellar tissues were allowed to reach cell confluency and were kept for three passages before cell proliferative analysis in the presence of human serum HS or fetal bovine serum FBS.

For colony-forming unit CFU quantification, a clonogenic assay was performed Figure 2 a. The colonies formed from the suprapatellar SVF were not only more abundant, but they were always bigger Figure 2 aright panel and exhibited a faster proliferative profile.

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In fact, when the ASC were cultured with either FBS or HS, the decret rates, in terms of the number of cells per growth dwcreto, within a cell growth curve daily analysis, were significantly higher from those cells amplified from the suprapatellar fat tissue compared with infrapatellar.

The differential growth rate was significantly different eight days after culture in the presence of HS and ten days after culture in FBS Figure 2 b. The ultrastructural analysis of the suprapatellar cell cultures grown in FBS or HS showed higher inclusion decreti accumulation indicated by white arrows and higher distribution of intermediate filaments indicated by black arrows in the presence of HS-containing medium Figure 2 c. Supra- and infrapatellar ASC proliferative activity.

The generated colonies were counted after Giemsa staining right panel. To further assess the stem cell characteristics from both adipose tissue sources, the supra- and infrapatellar fat pads, we compared the differentiation multipotency between both samples Figure 3. Both underwent induced differentiation into the adipogenic, osteogenic, and chondrogenic lineages Figure 3. However, the extent of differentiation was not identical. Suprapatellar-derived ASC showed higher osteogenic and chondrogenic potential in comparison with the infrapatellar-derived and infrapatellar-differentiated cells as indicated by quantification decretk Alizarin red and Alcian blue staining, respectively Figure 3right panels.

The higher chondrogenic potential of the suprapatellar-derived cells was also supported by the increased expression of Sox9 greena chondrocyte precursor marker [ 29 ].

This differential multipotency yields the suprapatellar fat pad a promising cell source for cartilage tissue repair. Multilineage potential of suprapatellar- and infrapatellar-derived ASC. All directed differentiation processes were induced from both samples, suprapatellar- and infrapatellar-derived ASC at passages after 4.

To quantify the efficiency of cell differentiation, specific staining was employed for each process: In addition, immunodetection of Connexin 43 red; osteocyte marker and phalloidin green; cytoskeletal marker was performed to evaluate osteocyte maturation.

Sox9 green counterstained with DAPI blue, nucleus stained newly generated chondrocytes. To assess whether the increased cell proliferative and cell differentiation properties shown in vitro of the suprapatellar-derived MSCs would be functionally efficient in cartilage regeneration, in vivo cell transplantation assay in a severe OA mouse model was performed.

Five days after injection, when total cartilage destruction occurs previously evaluated by histological analysis; data not shown10 5 hASC or the SVF or growth medium control was injected into the intra-articular space. It has been shown that PRP infiltration, increasingly implemented in regular clinical practice, can reduce decretoo and improve joint function with a notable improvement on the quality of life of patients [ 3031 ]. Therefore, PRP was also injected in an additional group of animals PRP in order to compare the regenerative effectiveness on cell transplantation in comparison with growth factor infiltration Figure 4.

Functional and structural cartilage regeneration. Representative images of a frontal section stained with safranin-O are shown vecreto each group. All cell nuclei were detected with DAPI staining blue.

Once a week after treatments, the volume of the knees was measured using an automatic caliper. As shown in Figure 4 aa significant increase in knee size occurs in all three groups after OA induction during the whole experiment. This result indicates a significant anti-inflammatory effect of the treatments.

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However, when we examined the structural regenerative effect by quantifying GAG Figure 4 b and the OA damage score Figure 4 cwe observed a unique consistent structural regeneration of the loss of cartilage when the animals were treated with hASC in comparison with the control group Figures 4 b and 4 c.

A representative safranin-O staining image for each treated group showed a complete absence of articular cartilage in the control animal and safranin-O-positive areas compatible with regenerating cartilage more prominent in the hASC-treated animal Figure 4 d.

To explore the contribution of the ectopic ASC to the regeneration of the articular cartilage, we evaluated the induced chondrogenic activity by the detection of Sox9 a chondrocyte progenitor marker positive cells in the control and supra-hASC-treated joints Figure secreto e.

As shown in the representative images, an increase in Sox9 positive cells was detected in the hASC group Figure 4 e. However, few of the Sox9 positive cells costained with the specific immunodetection for human cells antinuclei; data not shown indicating an endogenous 212 of the cartilage repair by the hASC transplantation.