Formulation and optimization of polymeric nanoparticles loaded with riolozatrione: a promising nanoformulation with potential antiherpetic activity.

Riolozatrione (RZ) is a diterpenoid compound isolated from a dichloromethane extract of the Jatropha dioica root. This compound has been shown to possess moderate antiherpetic activity in vitro. However, because of the poor solubility of this compound in aqueous vehicles, generating a stable formulation for potential use in the treatment of infection is challenging. The aim of this work was to optimize and physio-chemically characterize Eudragit® L100-55-based polymeric nanoparticles (NPs) loaded with RZ (NPR) for in vitro antiherpetic application. The NPs formulation was initially optimized using the dichloromethane extract of J. dioica, the major component of which was RZ. The optimized NPR formulation was stable, with a size of 263 nm, polydispersity index < 0.2, the zeta potential of -37 mV, and RZ encapsulation efficiency of 89 %. The NPR showed sustained release of RZ for 48 h with release percentages of 95 and 97 % at neutral and slightly acidic pH, respectively. Regarding in vitro antiherpetic activity, the optimized NPR showed a selectivity index for HSV-1 of ≈16 and for HSV-2 of 13.

Effect of Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Application in Achilles-Tendon Injury in an Animal Model.

Background: Achilles-tendon rupture prevails as a common tendon pathology. Adipose-derived mesenchymal stem cells (ADMSCs) are multipotent stem cells derived from adipose tissue with attractive regeneration properties; thus, their application in tendinopathies could be beneficial. Methods: Male rabbit ADMSCs were obtained from the falciform ligament according to previously established methods. After tenotomy and suture of the Achilles tendon, 1 × 106 flow-cytometry-characterized male ADMSCs were injected in four female New Zealand white rabbits in the experimental group (ADMSC group), whereas four rabbits were left untreated (lesion group). Confirmation of ADMSC presence in the injured site after 12 weeks was performed with quantitative sex-determining region Y (SRY)-gene RT-PCR. At Week 12, histochemical analysis was performed to evaluate tissue regeneration along with quantitative RT-PCR of collagen I and collagen III mRNA. Results: Presence of male ADMSCs was confirmed at Week 12. No statistically significant differences were found in the histochemical analysis; however, statistically significant differences between ADMSC and lesion group expression of collagen I and collagen III were evidenced, with 36.6% and 24.1% GAPDH-normalized mean expression, respectively, for collagen I (p < 0.05) and 26.3% and 11.9% GAPDH-normalized mean expression, respectively, for collagen III (p < 0.05). The expression ratio between the ADMSC and lesion group was 1.5 and 2.2 for collagen I and collagen III, respectively. Conclusion: Our results make an important contribution to the understanding and effect of ADMSCs in Achilles-tendon rupture.

Pilot study: genetic distribution of AR, FGF5, SULT1A1 and CYP3A5 polymorphisms in male Mexican population with androgenetic alopecia.

Genetics is responsible for 80% of androgenetic alopecia (AGA) predisposition. Several single nucleotide polymorphisms (SNPs) have been linked to AGA risk and the metabolism of its first-line therapies. Genotypic and allelic frequencies have not been described in Mexican individuals; therefore, the aim of this study was to describe the genetic distribution of SNPs associated with AGA predisposition and drug metabolism. Using Real Time-PCR, we genotyped SNPs rs4827528 (AR), rs7680591 (FGF5), rs1042028, rs1042157, rs788068 and rs6839 (SULT1A1) and rs776746 (CYP3A5) in 125 (controls = 60, cases = 65) male volunteers from Northern and Western Mexico. The SULT1A1 SNPs rs1042028 (C/T) and rs788068 (T/A/C) resulted in a 100% distribution of the ancestral allele C and mutated allele A, respectively; rs1042028 diverges from the previously reported frequency, while the rs788068 ancestral allele was found to be more predominant than the reported frequency. Rs1042028, rs788068 and rs4827528, were not in Hardy-Weinberg (HW) equilibrium; conversely, rs1042157 and rs6839, rs776746, and rs7680591 followed HW principles. A statistically significant difference (P<0.05) was obtained for the rs1042157 allelic frequency between cases and controls in Western Mexico. We reported the genotypic and allelic frequencies of seven polymorphisms in Mexican individuals from Northern and Western Mexico.

Sequential growth factor exposure of human Ad-MSCs improves chondrogenic differentiation in an osteochondral biphasic implant.

Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFß1 (10 ng/ml) and a final step of exposure to TGFß1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.

A Cellularized Biphasic Implant Based on a Bioactive Silk Fibroin Promotes Integration and Tissue Organization during Osteochondral Defect Repair in a Porcine Model.

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Advantages of adipose tissue stem cells over CD34+ mobilization to decrease hepatic fibrosis in Wistar rats.

INTRODUCTION AND OBJECTIVES: Chronic liver inflammation may lead to hepatic cirrhosis, limiting its regenerative capacity. The clinical standard of care is transplantation, although stem cell therapy may be an alternative option. The study aim was to induce endogenous hematopoietic stem cells (HSCs) with granulocyte colony stimulating factor (G-CSF) and/or intravenous administration of adipose tissue-derived mesenchymal stem cells (MSCs) to decrease hepatic fibrosis in an experimental model. MATERIAL AND METHODS: A liver fibrosis model was developed with female Wistar rats via multiple intraperitoneal doses of carbon tetrachloride. Three rats were selected to confirm cirrhosis, and the rest were set into experimental groups to evaluate single and combined therapies of G-CSF-stimulated HSC mobilization and intravenous MSC administration. RESULTS: Treatment with MSCs and G-CSF significantly improved alanine amino transferase levels, while treatment with G-CSF, MSCs, and G-CSF+MSCs decreased aspartate amino transferase levels. Hepatocyte growth factor (HGF) and interleukin 10 levels increased with MSC treatment. Transforming growth factor ß levels were lower with MSC treatment. Interleukin 1ß and tumor necrosis factor alpha levels decreased in all treated groups. Histopathology showed that MSCs and G-CSF reduced liver fibrosis from F4 to F2. CONCLUSIONS: MSC treatment improves liver function, decreases hepatic fibrosis, and plays an anti-inflammatory role; it promotes HGF levels and increased proliferating cell nuclear antigen when followed by MSC treatment mobilization using G-CSF. When these therapies were combined, however, fibrosis improvement was less evident.

A Bioactive Cartilage Graft of IGF1-Transduced Adipose Mesenchymal Stem Cells Embedded in an Alginate/Bovine Cartilage Matrix Tridimensional Scaffold.

Articular cartilage injuries remain as a therapeutic challenge due to the limited regeneration potential of this tissue. Cartilage engineering grafts combining chondrogenic cells, scaffold materials, and microenvironmental factors are emerging as promissory alternatives. The design of an adequate scaffold resembling the physicochemical features of natural cartilage and able to support chondrogenesis in the implants is a crucial topic to solve. This study reports the development of an implant constructed with IGF1-transduced adipose-derived mesenchymal stem cells (immunophenotypes: CD105+, CD90+, CD73+, CD14-, and CD34-) embedded in a scaffold composed of a mix of alginate/milled bovine decellularized knee material which was cultivated in vitro for 28 days (3CI). Histological analyses demonstrated the distribution into isogenous groups of chondrocytes surrounded by a de novo dense extracellular matrix with balanced proportions of collagens II and I and high amounts of sulfated proteoglycans which also evidenced adequate cell proliferation and differentiation. This graft also shoved mechanical properties resembling the natural knee cartilage. A modified Bern/O'Driscoll scale showed that the 3CI implants had a significantly higher score than the 2CI implants lacking cells transduced with IGF1 (16/18 vs. 14/18), representing high-quality engineering cartilage suitable for in vivo tests. This study suggests that this graft resembles several features of typical hyaline cartilage and will be promissory for preclinical studies for cartilage regeneration.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer.

INTRODUCTION: Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-ß1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. METHODS: Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-ß1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. RESULTS: Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. CONCLUSION: Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair.

Human bone morphogenetic protein 2-transduced mesenchymal stem cells improve bone regeneration in a model of mandible distraction surgery.

BACKGROUND: Bone morphogenetic proteins (BMPs) are actively involved in ossification, and BMP-2 participates throughout the entire process. Gene therapy for bone regeneration using adenovirus-expressing BMPs has been successful in small mammals, but it has not been satisfactory in large mammals. METHODS: We generated a 3-component implant (3C graft) comprising autologous mesenchymal stem cells (MSCs), ex vivo transduced with an adenovirus vector-expressing BMP-2 and embedded in a demineralized human bone matrix (DBM). RESULTS: In vitro studies demonstrated vector-induced osteogenesis; osteoblast population and mineralization of the extracellular matrix were greater in the vector-transduced cultures than in the controls (nontransduced MSCs stimulated with osteogenic media were used as positive controls, and nontransduced MSCs served as a negative control). The 3-component grafts were used to fill osteotomies created by bone distraction surgery in mongrel dogs. Control groups comprised dogs with bone distraction alone and dogs with nontransduced MSC grafts. The radiography follow-up, performed 10 weeks after distraction, demonstrated a remarkable reduction in the consolidation period compared with controls. Postmortem mandibles submitted for anatomic and histologic analyses showed improved remodeling and bone maturation in the 3C-grafted dogs. Inflammatory infiltrates were not observed in any of the treated areas, and no liver toxicity was detected. CONCLUSIONS: We demonstrated acceleration of osteogenesis in a dog model for bone distraction by using an implant of BMP-2 modified MSCs. These results are helpful for future clinical trials of mandible bone distraction.