Ex Vivo Maturation of 3D-Printed, Chondrocyte-Laden, Polycaprolactone-Based Scaffolds Prior to Transplantation Improves Engineered Cartilage Substitute Properties and Integration.

OBJECTIVE: The surgical management of nasal septal defects due to perforations, malformations, congenital cartilage absence, traumatic defects, or tumors would benefit from availability of optimally matured septal cartilage substitutes. Here, we aimed to improve in vitro maturation of 3-dimensional (3D)-printed, cell-laden polycaprolactone (PCL)-based scaffolds and test their in vivo performance in a rabbit auricular cartilage model. DESIGN: Rabbit auricular chondrocytes were isolated, cultured, and seeded on 3D-printed PCL scaffolds. The scaffolds were cultured for 21 days in vitro under standard culture media and normoxia or in prochondrogenic and hypoxia conditions, respectively. Cell-laden scaffolds (as well as acellular controls) were implanted into perichondrium pockets of New Zealand white rabbit ears (N = 5 per group) and followed up for 12 weeks. At study end point, the tissue-engineered scaffolds were extracted and tested by histological, immunohistochemical, mechanical, and biochemical assays. RESULTS: Scaffolds previously matured in vitro under prochondrogenic hypoxic conditions showed superior mechanical properties as well as improved patterns of cartilage matrix deposition, chondrogenic gene expression (COL1A1, COL2A1, ACAN, SOX9, COL10A1), and proteoglycan production in vivo, compared with scaffolds cultured in standard conditions. CONCLUSIONS: In vitro maturation of engineered cartilage scaffolds under prochondrogenic conditions that better mimic the in vivo environment may be beneficial to improve functional properties of the engineered grafts. The proposed maturation strategy may also be of use for other tissue-engineered constructs and may ultimately impact survival and integration of the grafts in the damaged tissue microenvironment.

Composite alginate-gelatin hydrogels incorporating PRGF enhance human dental pulp cell adhesion, chemotaxis and proliferation.

The increasing prevalence of tissue injuries is fueling the development of autologous biological treatments for regenerative medicine. Here, we investigated the potential of three different bioinks based on the combination of gelatin and alginate (GA), enriched in either hydroxyapatite (GAHA) or hydroxyapatite and PRGF (GAHAP), as a favorable microenvironment for human dental pulp stem cells (DPSCs). Swelling behaviour, in vitro degradation and mechanical properties of the matrices were evaluated. Morphological and elemental analysis of the scaffolds were also performed along with cytocompatibility studies. The in vitro cell response to the different scaffolds was also assessed. Results showed that all scaffolds presented high swelling capacity, and those that contained HA showed higher Young's modulus. GAHAP had the lowest degradation rate and the highest values of cytocompatibility. Cell adhesion and chemotaxis were significantly increased when PRGF was incorporated to the matrices. GAHA and GAHAP compositions promoted the highest proliferative rate as well as significantly stimulated osteogenic differentiation. In conclusion, the enrichment with PRGF improves the regenerative properties of the composites favouring the development of personalized constructs.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

Three-Dimensional Bioprinting Scaffolding for Nasal Cartilage Defects: A Systematic Review.

BACKGROUND: In recent years, three-dimensional (3D)-printing of tissue-engineered cartilaginous scaffolds is intended to close the surgical gap and provide bio-printed tissue designed to fit the specific geometric and functional requirements of each cartilage defect, avoiding donor site morbidity and offering a personalizing therapy. METHODS: To investigate the role of 3D-bioprinting scaffolding for nasal cartilage defects repair a systematic review of the electronic databases for 3D-Bioprinting articles pertaining to nasal cartilage bio-modelling was performed. The primary focus was to investigate cellular source, type of scaffold utilization, biochemical evaluation, histological analysis, in-vitro study, in-vivo study, animal model used, length of research, and placement of experimental construct and translational investigation. RESULTS: From 1011 publications, 16 studies were kept for analysis. About cellular sources described, most studies used primary chondrocyte cultures. The cartilage used for cell isolation was mostly nasal septum. The most common biomaterial used for scaffold creation was polycaprolactone alone or in combination. About mechanical evaluation, we found a high heterogeneity, making it difficult to extract any solid conclusion. Regarding biological and histological characteristics of each scaffold, we found that the expression of collagen type I, collagen Type II and other ECM components were the most common patterns evaluated through immunohistochemistry on in-vitro and in-vivo studies. Only two studies made an orthotopic placement of the scaffolds. However, in none of the studies analyzed, the scaffold was placed in a subperichondrial pocket to rigorously simulate the cartilage environment. In contrast, scaffolds were implanted in a subcutaneous plane in almost all of the studies included. CONCLUSION: The role of 3D-bioprinting scaffolding for nasal cartilage defects repair is growing field. Despite the amount of information collected in the last years and the first surgical applications described recently in humans. Further investigations are needed due to the heterogeneity on mechanical evaluation parameters, the high level of heterotopic scaffold implantation and the need for quantitative histological data.

Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis.

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients' health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium-supplemental hormone epithelial medium (SHEM)-supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.

Recent advances in gelatin-based therapeutics.

INTRODUCTION: Biomaterials have provided a wide range of exciting opportunities in tissue engineering and regenerative medicine. Gelatin, a collagen-derived natural biopolymer, has been extensively used in regenerative medicine applications over the years, due to its cell-responsive properties and the capacity to deliver a wide range of biomolecules. AREAS COVERED: The most relevant properties of gelatin as biomaterial are presented together with its main therapeutic applications. The latter includes drug delivery systems, tissue engineering approaches, potential uses as ink for 3D/4D Bioprinting, and its relevance in organ-on-a-chip platforms. EXPERT OPINION: Advances in polymer chemistry, mechanobiology, imaging technologies, and 3D biofabrication techniques have expanded the application of gelatin in multiple biomedical research applications ranging from bone and cartilage tissue engineering, to wound healing and anti-cancer therapy. Here, we highlight the latest advances in gelatin-based approaches within the fields of biomaterial-based drug delivery and tissue engineering together with some of the most relevant challenges and limitations.

Hyaluronic Acid Combined with Serum Rich in Growth Factors in Corneal Epithelial Defects.

The aim of this study is to assess if an adhesive biopolymer, sodium hyaluronate (NaHA), has synergistic effects with s-PRGF (a serum derived from plasma rich in growth factors and a blood derivative that has already shown efficacy in corneal epithelial wound healing), to reduce time of healing or posology. In vitro proliferation and migration studies, both in human corneal epithelial (HCE) cells and in rabbit primary corneal epithelial (RPCE) cultures, were carried out. In addition, we performed studies of corneal wound healing in vivo in rabbits treated with s-PRGF, NaHA, or the combination of both. We performed immunohistochemistry techniques (CK3, CK15, Ki67, ß4 integrin, ZO-1, α-SMA) in rabbit corneas 7 and 30 days after a surgically induced epithelial defect. In vitro results show that the combination of NaHA and s-PRGF offers the worst proliferation rates in both HCE and RPCE cells. Addition of NaHA to s-PRGF diminishes the re-epithelializing capability of s-PRGF. In vivo, all treatments, given twice a day, showed equivalent efficacy in corneal epithelial healing. We conclude that the combined use of s-PRGF and HaNA as an adhesive biopolymer does not improve the efficacy of s-PRGF alone in the wound healing of corneal epithelial defects.

Advances in stem cell therapy for cartilage regeneration in osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is a progressive joint disease that compromises the structural integrity of cartilage tissue. Conventional treatments based on medication or surgery are nowadays inefficient and cell-based therapy has emerged as one of the most promising methods for cartilage regeneration. The first therapy developed for cartilage defects was autologous chondrocyte implantation, but in the last few decades stem cells (SCs) from different sources have been proposed as a possible alternative for OA. AREAS COVERED: SC sources and available delivery procedures (scaffolds/hydrogels) are presented, along with the main issues arisen in this regard. Thereafter, preclinical and clinical trials performed in recent years are reviewed in order to take a glance toward the potential benefits that such therapies could deliver to the patients. EXPERT OPINION: SCs have proven their potential and safety for OA treatment. Nevertheless, there are still many questions to be resolved before their widespread used in clinical practice, such as the treatment mechanism, the best cell source, the most appropriate processing method, the most effective dose and delivery procedure, and their efficacy. In this sense, long-term follow-up and larger randomized controlled trials utilizing standardized and established outcome scores are mandatory to make objective conclusions.

Serum from plasma rich in growth factors regenerates rabbit corneas by promoting cell proliferation, migration, differentiation, adhesion and limbal stemness.

PURPOSE: To evaluate the regenerating potential and the mechanisms through which the autologous serum derived from plasma rich in growth factors (s-PRGF) favours corneal wound healing in vitro and in vivo. METHODS: We compared the effect of various concentrations of s-PRGF versus fetal bovine serum (FBS) and control treatment in rabbit primary corneal epithelial and stromal cells and wounded rabbit corneas. Cell proliferation was measured using an enzymatic colorimetric assay. In vitro and in vivo wound-healing progression was assessed by image-analysis software. Migration and invasion were evaluated using transfilter assays. Histological structure was analysed in stained sections. Protein expression was evaluated by immunohistochemistry. RESULTS: s-PRGF promoted the robust proliferation of epithelial cultures at any concentration, similar to FBS. Likewise, s-PRGF and FBS produced similar re-epithelialization rates in in vitro wound-healing assays. In vivo, s-PRGF treatment accelerated corneal wound healing in comparison with control treatment. This difference was significant only for 100% s-PRGF treatment in our healthy rabbit model. Histological analysis confirmed normal epithelialization in all cases. Immunohistochemistry showed a higher expression of cytokeratins 3/76 and 15, zonula occludens-1 and alpha-smooth muscle actin proteins as a function of s-PRGF concentration. Notably, keratocyte density in the anterior third of the stroma increased with increase in s-PRGF concentration, suggesting an in vivo chemotactic effect of s-PRGF on keratocytes that was further confirmed in vitro. CONCLUSION: s-PRGF promotes proliferation and migration and influences limbal stemness, adhesion and fibrosis during corneal healing.

Corneal wound healing promoted by 3 blood derivatives: an in vitro and in vivo comparative study.

PURPOSE: The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]. METHODS: Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment. RESULTS: In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment. CONCLUSIONS: All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.