Francisco Muruzabal

Ligamentization of Tendon Grafts Treated With an Endogenous Preparation Rich in Growth Factors: Gross Morphology and Histology

PURPOSE To investigate whether the application of a particular platelet-rich plasma preparation rich in growth factors (PRGF) during anterior cruciate ligament (ACL) surgery gives a potential advantage for better tendon graft ligamentization. METHODS This study included 37 volunteers who underwent either conventional (control group, n = 15) or PRGF-assisted (n = 22) ACL reconstruction with an autogenous hamstring and required second-look arthroscopy to remove hardware or loose bodies, treat meniscal tears or plica syndrome, or resect cyclops lesions at 6 to 24 months after ACL surgery. The gross morphologies of the grafts were evaluated on second-look arthroscopy by use of the full arthroscopic score (0 to 4 points) to evaluate graft thickness and apparent tension (0 to 2 points) plus synovial coverage (0 to 2 points). At the same time, biopsy specimens were harvested uniformly from the grafted tendons. In these specimens the histologic transformation of the tendon graft to ACL-like tissue was evaluated by use of the Ligament Tissue Maturity Index, and a score to assess the progression of new connective tissue enveloping the graft was created by use of 3 criteria previously used to characterize changes during ligament healing: cellularity, vascularity, and collagen properties. RESULTS The overall arthroscopic evaluation of PRGF-treated grafts showed an excellent rating in 57.1% of the knees (score of 4) and a fair rating in 42.9% (score of 2 or 3). In contrast, evaluation of untreated grafts showed an excellent rating in 33.3% of the knees, a fair rating in 46.7%, and a poor rating in 20% (score of 0 or 1). Overall, arthroscopic evaluations were not statistically different between PRGF and control groups (P = .051). PRGF treatment influenced the histologic characteristics of the tendon graft, resulting in tissue that was more mature than in controls (P = .024). Histologically evident newly formed connective tissue enveloping the graft was present in 77.3% of PRGF-treated grafts and 40% of controls. The appearance of the connective tissue envelope changed with increasing time from surgery. On the basis of the histologic findings, we suggest that the remodeling of PRGF-treated grafts involves the formation of synovial-like tissue enveloping the graft. This tissue is eventually integrated in the remodeled tendon graft, conferring a similar appearance to the normal ACL. CONCLUSIONS The use of PRGF influenced the histologic characteristics of tendon grafts, resulting in more remodeling compared with untreated grafts. We have shown temporal histologic changes during the 6- to 24-month postoperative period of graft maturation, with newly formed connective tissue enveloping most grafts treated with PRGF. LEVEL OF EVIDENCE Level III, case-control study.

Immunocytochemical detection of leptin in non-mammalian vertebrate stomach

Leptin is a hormone produced and secreted mainly by adipocytes, but also by other tissues such as placenta, brain, mammary, and pituitary glands. The gastric epithelium has also been reported as a source of leptin in mammals. In this study we examined the presence of leptin in the stomach of non-mammalian vertebrates (trout, frog, lizard, and snake). Immunolabeling for leptin was found in the oxyntic-peptic cells of the frog and the two reptilian species studied, but not in the trout. In the trout and the lizard leptin immunoreactivity was also detected in scattered cells presenting the typical features of endocrine cells. In the trout, the frog and the snake, in addition to the epithelium, leptin immunostain was found in elements of the enteric nervous system that were also positive for VIP.

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis.

BackgroundThe recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear.MethodsThe proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 μm in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence.ResultsSpheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells.Conclusion3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

Leptin expression in the rat ovary depends on estrous cycle.

Leptin is a hormone originally identified in adipocytes. It is involved in the regulation of fat deposition and energy expenditure and in other functions, such as reproduction. The presence of leptin has been reported in several reproductive organs. However, few studies have addressed its expression in the ovary. Moreover, the existing information is not consistent with regard to the particular cell types responsible for leptin expression. In this work we studied the distribution of leptin in the rat ovary by immunohistochemistry (IHC) and in situ hybridization (ISH). Leptin staining was found in steroid-producing cells: thecal, luteal, and interstitial cells. The strongest signal with both techniques was found in the cytoplasm of oocytes. A weak reaction for leptin mRNA was detected in granulosa of all growing follicles, although leptin protein was found only in the mature follicle. Western blotting analysis detects a strongly reactive 16-kD band, giving further support to the presence of leptin in the rat ovary. Variations in this immunoreactive band were found throughout the estrous cycle. Localization of leptin in the ovary may contribute to a better understanding of female reproductive function.

Autologous serum and plasma rich in growth factors in ophthalmology: preclinical and clinical studies.

The use of blood derivatives represents an alternative therapeutic approach that is gaining interest in regenerative medicine due to its potential to stimulate and accelerate tissue healing. Autologous serum eye drops and platelet‐enriched plasma eye drops are being used in the treatment of different ophthalmological disorders. In this review, we summarize the different blood‐derived formulations used in the treatment and care of ocular surface disorders. The biological basis and use of autologous serum and plasma rich in growth factors are deeply evaluated as well as the challenges to be addressed in the future in this new generation of blood‐derived therapies.

Reduced adipose tissue mass and hypoleptinemia in iNOS deficient mice: effect of LPS on plasma leptin and adiponectin concentrations

The aim of this study was to evaluate the impact of the lack of inducible NO synthase (iNOS) on body weight and adipose tissue mass as well as on plasma leptin and adiponectin in basal conditions and 6 h after lipopolysaccharide (LPS) administration in mice. Body weight was not different among male, six‐week‐old wild‐type (WT) and iNOS−/− animals. However, the amount of epididymal white adipose tissue (EWAT) in iNOS−/− mice was significantly reduced (P<0.05). Circulating leptin and leptin mRNA in EWAT were decreased in iNOS−/− mice (P<0.05 and P<0.01, respectively). Plasma adiponectin and adiponectin mRNA were unchanged. LPS administration increased plasma leptin in both genotypes (P<0.05). Neither genotype nor treatment changed plasma adiponectin. In summary, iNOS−/− mice exhibited normal body weight but reduced adipose mass accompanied by hypoleptinemia. Leptin responsiveness to LPS in iNOS−/− mutants is preserved, showing that the LPS‐induced rise in leptin is independent of the presence of functional iNOS. In addition, iNOS deficiency or LPS does not influence expression and circulating levels of adiponectin.

The use of plasma rich in growth factors (PRGF-Endoret) in the treatment of a severe mal perforant ulcer in the foot of a person with diabetes

A 71 year old person with diabetes with a severe mal perforant ulcer in the right foot was treated twice with autologous plasma-rich in growth factors (PRGF) obtained from her own blood. After PRGF treatment the severe mal perforant ulcer completely healed in 10 weeks.

Plasma rich in growth factors (PRGF) eye drops stimulates scarless regeneration compared to autologous serum in the ocular surface stromal fibroblasts

Autologous serum (AS) eye drops was the first blood-derived product used for the treatment of corneal pathologies but nowadays PRGF arises as a novel interesting alternative to this type of diseases. The purpose of this study was to evaluate and compare the biological outcomes of autologous serum eye drops or Plasma rich in growth factors (PRGF) eye drops on corneal stromal keratocytes (HK) and conjunctival fibroblasts (HConF). To address this, blood from healthy donors was collected and processed to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Blood-derivates were aliquoted and stored at -80°C until use. PDGF-AB, VEGF, EGF, FGFb and TGF-β1 were quantified. The potential of PRGF and AS in promoting wound healing was evaluated by means of proliferation and migration assays in HK and HConF. Fibroblast cells were induced to myofibroblast differentiation after treatment with 2.5ng/mL of TGF-β1. The capability of PRGF and AS to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results showed significant higher levels of all growth factors analyzed in PRGF eye drops compared to AS. Moreover, PRGF eye drops enhanced significantly the biological outcomes of both HK and HConF, and reduced TGF-β1-induced myofibroblast differentiation in contrast to autologous serum eye drops (AS). In summary, these results suggest that PRGF exerts enhanced biological outcomes than AS. PRGF may improve the treatment of ocular surface wound healing minimizing the scar formation compared to AS. Results obtained herein suggest that PRGF protects and reverses the myofibroblast phenotype while promotes cell proliferation and migration.

Biological Stability of Plasma Rich in Growth Factors Eye Drops After Storage of 3 Months.

Purpose: We evaluated whether plasma rich in growth factors eye drops maintain their composition and biological activity after storage for 3 months at −20°C and after storage at 4°C or room temperature (RT) for 24 hours, compared with samples obtained at time 0 (fresh samples). Methods: Blood from 10 healthy donors was collected, centrifuged, and plasma rich in growth factors was prepared by avoiding the collection of the buffy coat. Eye drops were kept fresh or were stored at −20°C for 15, 30, and 90 days. For each time, 2 aliquots were kept at RT or at 4°C for 24 hours. Osmolarity, vitamin A, fibronectin, platelet-derived growth factor-AB, vascular endothelial growth factor, epithelial growth factor, and transforming growth factor-&bgr;1 were quantified. The proliferative and migratory potential of the eye drops was assayed on primary human keratocytes. Results: Platelet-derived growth factor-AB, vascular endothelial growth factor, epithelial growth factor, and vitamin A levels remained constant for each time and for each storage condition, whereas fibronectin, transforming growth factor-&bgr;1, and osmolarity values were slightly modified after freezing. Cell proliferation and migration were significantly enhanced with the biological eye drops independently of the time and the storage condition. No microbial contamination was observed in any plasma rich in growth factors eye drops. Conclusions: Plasma rich in growth factors eye drops can be stored for up to 3 months without any reduction of the main proteins involved in ocular surface healing. Their use during 24 hours either at 4°C or at RT did not alter the composition and the in vitro biological activity of the eye drops.

Implementation of a more physiological plasma rich in growth factor (PRGF) protocol: Anticoagulant removal and reduction in activator concentration

Abstract Plasma rich in growth factors (PRGF) is a biological therapy that uses patient’s own growth factors for promoting tissue regeneration. Given the current European regulatory framework in which anticoagulant solution in blood extraction tubes could be considered as a medicinal product, a new PRGF protocol has been developed. The actual protocol (PRGF-A) and the new one (PRGF-B) have been performed and compared under Good Laboratory Practices. PRGF-A protocol uses extraction tubes with 0.9 mL of trisodium citrate as anticoagulant and 50 μL of calcium chloride/mL PRGF to activate it. The PRGF-B reduces the amount of sodium citrate and calcium chloride to 0.4 mL and to 20 μL, respectively. Basic hematological parameters, platelet function, the scaffold obtaining process, growth factors content, and the biological effect were compared between both PRGF obtaining protocols. Results: PRGF-B protocol led to a statistically significant higher enrichment and recovery of platelets regarding to the PRGF-A. Hypotonic stress response by platelets was significantly better in the new protocol. A statistically significant decrease in the basal platelet activation status of PRGF-B compared to PRGF-A was also observed. The duration of the lag phase in the platelet aggregation assay was statistically lower for the PRGF-B protocol. Both the clotting and the clot retraction time were significantly reduced in the B protocol. A higher growth factor concentration was detected in the plasma obtained using the PRGF-B protocol. The new PRGF obtaining protocol, with a reduction in the amount of anticoagulant and activator, has even improved the actual one.