Descemet's membrane is the specialized extracellular matrix located between corneal stroma and endothelium. This basement membrane provides the biomechanical cues that sustain endothelium viability and function, making it an optimal cell scaffold. The present work describes a new decellularization protocol to remove the cellular components and obtain an acellular scaffold from Descemet's membrane. To induce cell lysis and eliminate all cytoplasmic and nuclear material, Descemet's membranes isolated from donor corneas were subjected to osmotic (hypotonic) shock. The efficiency of the decellularization process was evaluated by the quantification of total residual DNA and analysis by gel electrophoresis. Nuclei removal and extracellular matrix integrity after treatment were verified by histological analysis. In particular, the maintenance of collagen, glycoproteins, perlecan and elastin was analyzed in decellularized tissues compared to untreated controls. DNA quantification showed a 99% reduction of total DNA amount in decellularized Descemet's membranes compared to control ones, with only 28.9 ± 9.86ng DNA/mg dry tissue residual. Furthermore, the agarose gel electrophoresis and the absence of visible nuclei after decellularization confirmed the efficiency of the process. Histological analyses showed that the composition of the extracellular matrix was not modified by the process. The decellularization protocol is effective in obtaining a Descemet's membrane that is depleted of donor DNA. Furthermore, the treatment preserves tissue matrix components. Descemet's membrane is already prestripped and provided by eye banks; therefore, a decellularized Descemet's membrane represents a valid candidate as a safe scaffold for intraocular surgery as, for example, in the treatment of refractory macular holes.

Bone morphogenetic protein (BMP) stimulated osteoinduction is critical for bone regeneration. Human demineralized bone matrix (DBM) has been one of the most widely used bone graft substitutes, but its osteoinductive potential is weak and clinical effectiveness limited in part due to ineffective processing methods. Here, we describe a novel process designed to enhance allograft bioactivity by increasing the bioavailability of the native BMPs present within the matrix the product of which we call Natural Matrix Protein® (NMP®). BMP-7 release was quantified from NMP and DBM prepared from both bovine and human cortical bone. In both species, NMP significantly increased BMP-7 levels in acidic and physiologic extracts compared to DBM. NMP also demonstrated sustained release of BMP-7 and total protein for up to 12 weeks in simulated body fluid. Osteoinductive potential was evaluated in vitro using C2C12 cells and osteoinductivity in vivo in the athymic rat model. Direct treatment of cells with NMP in vitro produced a greater than tenfold increase in alkaline phosphatase activity at 40 mg/mL. In vivo, human NMP showed increased osteoinductivity compared to human DBM histologically, and the recovered NMP explants had significantly more mineralized bone and a higher bone volume fraction compared to DBM and to Infuse® Bone Graft (105µg rhBMP-2 on an absorbable collagen sponge) as measured by microCT. These findings demonstrate that the novel NMP process reproducibly increases BMP-7 bioavailability, that NMP implants produce sustained BMP and protein release and have marked increase in osteoinductive activity.

To evaluate the treatment success and reversal rate of corneal allograft rejections in post-penetrating keratoplasty patients. Retrospective cohort study was performed in post-penetrating keratoplasty patients treated for corneal allograft rejection between September 2015 and August 2024 in a tertiary eye center in Debrecen, Hungary. Indication of keratoplasty, onset of the allograft rejection, best corrected visual acuity (BCVA), corneal transparency on slit lamp examination and applied treatment were recorded. In our institution's patient registry, 68 allograft rejection episodes were found. Before the rejection, all grafts were completely transparent. The rejection episodes occurred median 21months after surgery. Diagnosis and treatment took place median 5days after the onset of symptoms. Allograft rejection could be successfully reversed in 48 of the total 68 cases (70.6%). Treatment was adjusted individually and all patients received local treatment; 16 received only local and 52 received combined systemic and local corticosteroids resulting in a similar rejection reversal rate (13/16 vs. 35/52; p = 0.359). Comparing first (47) and repeat grafts (21), there were no significant differences between the treatment success rate (33/47 vs. 15/21; P = 1.000) and the frequency of combined local-plus-systemic treatment (34/47 vs. 18/21; p = 0.355). Before the rejection episodes, BCVA was 0.40 ± 0.30, which decreased following the rejection (0.28 ± 0.28; p < 0.001). This reduction in BCVA was observed in successfully treated cases as well (0.44 ± 0.28 vs. 0.38 ± 0.27; p = 0.008). Our dexamethasone-based local treatment demonstrated similar effectiveness in reversing corneal allograft rejections in patients underwent penetrating keratoplasty compared to data on prednisolone by other studies. However, systemic steroid augmentation might be needed more frequently when using topical dexamethasone.

Regeneration of articular cartilage disorders is one of the critical challenges in musculoskeletal medicine. Tissue engineering could represent a therapeutic option to support cartilage regeneration. Natural and biological materials are appropriate for fabricating tissue engineering scaffolds because of their similarity to natural tissues. The properties of amniotic membranes, including low immunogenicity, anti-inflammatory role, cell loading capability, expression of various growth factors, and chondroprotective effect, make them an interesting option for cartilage regeneration. This review studied the structure of articular cartilage and potential applications of the human amniotic membrane (AM) for articular cartilage regeneration. In addition, processing and decellularization methods of AM and the most common forms of amniotic membrane used in cartilage regeneration, including sheet, injectable form, and 3D forms, were studied. This review highlights the benefits of amniotic membrane applications in cartilage regeneration and clinical trial studies.

This study aimed to determine the most feasible perinatal tissue for Good Manufacturing Practice (GMP)-compliant banking of mesenchymal stromal-like cells (MSC-like cells). It was hypothesized that amniotic fluid collected during cesarean section would yield lower contamination rates and greater processing feasibility compared with other perinatal tissues. This prospective observational study was conducted at a tertiary university hospital and included 32 healthy term pregnancies. A total of 160 perinatal samples-amniotic fluid, amniotic membrane, umbilical cord, intact placenta, and placental fragments-were obtained. A validated feasibility scoring system evaluated material acquisition difficulty, transportation logistics, storage duration, and processing complexity. Samples were stratified by delivery mode (cesarean section vs. vaginal delivery) and collection timing (within vs. outside laboratory working hours). Stem cell isolation, sterility assessment, and immunophenotypic characterization were performed. Due to the absence of trilineage differentiation assays and maternal-fetal origin confirmation, the isolated cells were defined as MSC-like cells rather than definitive fetal MSCs. Statistical analyses were performed using chi-square and Mann-Whitney U tests (p < 0.05). Samples collected via cesarean section demonstrated significantly lower rates of blood contamination (25.8% vs. 60.0%, p < 0.001) and bacterial contamination (25.8% vs. 60.0%, p < 0.001) compared with those from vaginal deliveries. Amniotic fluid achieved the highest acquisition score, required no enzymatic digestion, and had the shortest median isolation time (45min). It exhibited the lowest overall contamination and was the most suitable source for GMP-oriented MSC-like cell processing. Conversely, intact placenta and placental fragments showed the highest contamination rates, longest enzymatic processing times, and greatest logistical burden. While collection timing affected storage duration and workflow continuity, tissue type and delivery mode were the dominant determinants of feasibility. Cesarean section-derived amniotic fluid appears to be the most practical, sterile, and processing-efficient perinatal source for GMP-adapted MSC-like cell banking within the evaluated parameters. These results support its prioritization in the development of standardized collection and processing protocols for perinatal stromal cell applications in regenerative medicine.

The current investigation aimed to exhibit the impact of lyophilization media of frozen spermatozoa on the fertility potential of buffalo spermatozoa as indicated by comet assay and ICSI. Semen specimens were centrifugated at 700 × g for 20min using percoll gradient (45-90%), double washed in Tyrode's albumen lactate pyruvate (TALP) and diluted in the lyophilization media (media 1, 2, 3 and 4), correspondingly. Cooling of the diluted sperm cells in vapor of liquid nitrogen. Frozen samples were instantly put into the lyophilizer (-55°C, pressure 0.001 Mbar). After 24h of lyophilization, the semen specimens were kept for three months at 4°C. Frozen-dried semen was re-hydrated at room temperature in of milli-Q water(100 µL). Comet assay results of the frozen-dried semen exhibited that the TCM medium exhibited the lowest % of DNA deterioration [6.17] and the superior % of embryonic developmental rate,while Tris-EDTA medium exhibited the highest % of DNA deterioration [13.09]. The lowest successful % of ICSI exhibited upon using EGTA and EDTA media. It could be concluded that ICSI of frozen-dried spermatozoa upon using TCM medium provides the highest % of embryonic expansion. Also, Tris-EGTA and Tris-EDTA media exhibited the lowest successful percent.

Deep and extensive thermal burns with concurrent inhalation injuries can be associated with a high mortality rate, especially among elderly patients. Injuries of this type require treatment in highly. specialized centers. Early excision and autografting are the standard of care for extensive, deep burns. To achieve a functionally and aesthetically satisfactory burn scar, allogeneic acellular dermal matrices (ADMs) can be used as co-grafts alongside autologous split-thickness skin grafts (STSGs). Additionally, the application of in vitro-cultured autologous keratinocytes and fibroblasts has been shown to accelerate burn wound healing. Allogeneic amnion transplantation can also be performed to promote healing at donor sites. This paper presents a case report of a 65year-old patient with thermal burns covering 26% total body surface area (TBSA) with third-degree burns affecting the thorax, abdomen, back, right shoulder, right elbow, and right thigh, as well as airway involvement. The patient underwent multistage surgical treatment, including deep excision of necrotic tissues. The wound was treated using a combination of ADM, free STSG, in vitro-cultured skin cells, and local negative pressure wound therapy (NPWT). Allogeneic amnion grafts were applied to the donor sites, which were used multiple times after healing. Healing progress was monitored using laser speckle contrast analysis (LASCA). Additionally, scar viscoelasticity, transepidermal water loss, melanin content, epidermal thickness, and temperature were examined post-healing. Selected skin parameters were also assessed using high-frequency ultrasound. The patient was discharged on day 77, having spent 41days in the surgical ward and 36days in the rehabilitation ward, with fully healed wounds. It is important to note that rehabilitation began on the first day of hospitalization. Follow-up visits documented gradual improvement in the evaluated scar parameters.

Platelet lysate is a derivative of platelet-rich plasma that is used as supplement for in vitro cell culture media. A variety of protocols for its preparation have been described. However, its potential use in the clinical setting has been poorly studied. In the present work, the effect of several protocols on cell proliferation has been comparatively analyzed. Additionally, the effect of exposing bone and vascular tissues to different concentrations of platelet lysate has also been analyzed. Human fibroblast-like cells were used to test preparation protocols. Thawed skull and artery fragments were incubated with platelet lysate and seeded as explants in culture plates. Cell growth was evaluated quantitatively in the first assay (cell count) and qualitatively in the second (presence of growing cell colonies). The presence of leukocytes in the raw material to obtain the platelet lysate was correlated with higher cell proliferation. In all cultures from arteries and 71.4% of those from bones, the presence of viable cells was detected. No statistically significant differences that correlated with the percentage of platelet lysate used during the post-thaw incubation were observed. The main findings of this study revealed that: there is a contribution of bioactive substances for cell growth by lymphocytes, incubation with platelet lysate had no significant activating effect on cells in thawed tissues, arteries stored in liquid nitrogen retained cell viability for long periods (over 5years), and cell viability in bones stored at -80°C decreased after 3months.

In tissue engineering, natural and synthetic nanofibers that can regenerate body damage have been successfully used in the repair of many lesion types, including peripheral neural lesions, in recent years. So, we developed three different nanofibers that we think can regenerate peripheral nerve damage. Three different nanofibers based on biodegradable poly-ε-caprolactone (PCL); Pure PCL (PCL) nanofiber, 70% PCL and 30% bioactive glass (PCL/BG) hybrid nanofiber, and 0.1% vitamin B12 added (PCL/BG)-B12 hybrid nanofiber were produced by electrospinning. Sol-gel method was used in the synthesis of biomaterials containing bioactive glass. The nanofibers were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and fourier transform infrared spectroscopy (FT-IR). Cell viability assays were performed with healthy L929 fibroblast cells and PC12 cells to evaluate the biocompatibility of nanofibers. Neuronal differentiation of PC12 cells were stimulated by nerve growth factor (NGF). To assess the differentiation levels of PC12 cells, the length of neurites and number of outgrowing neurites per cell was evaluated morphologically, and NGF production levels of the cells were determined by ELISA. The results suggest that these biocompatible nanofibers stimulated PC12 cell survival and neuronal differentiation. Among these scaffolds, PCL/BG-B12 nanofibers strikingly triggered NGF production of PC12 cells as a hallmark of neuroregeneration. Thus, the nanofibers are capable of neuroprotective properties due to their safe, supporting proliferation, and NGF-releasing capacity. Additionally, it could be suggested that the PCL/BG nanofiber and vitamin B12 have the potential to be used in further studies for neurodegenerative diseases.

Tissue engineering (TE) combines cells, biomaterials, and bioactive molecules to create functional tissue constructs aimed at restoring tissue function and improving patient outcomes. The human amniotic membrane (HAM) is a widely studied biological scaffold for various biomedical applications. Decellularization of HAM (dHAM) is necessary to reduce graft rejection but depletes stem cells and growth factors, potentially limiting regenerative potential. This study investigates the recellularization of dHAM with adipose-derived mesenchymal stem cells (AdMSCs) to enhance its bioactivity using a novel 3D seeding technique. Decellularized HAM (dHAM) was recellularized with AdMSCs employing a novel 3D seeding method to achieve uniform cell distribution within the scaffold. The viability, differentiation potential, and morphology of AdMSCs were assessed in both 2D and 3D culture systems. Flow cytometry was used to evaluate the differentiation capacity of AdMSCs into osteogenic, chondrogenic, and adipogenic lineages. Field emission scanning electron microscopy (FESEM) was utilized to analyze cell morphology and penetration depth within the scaffold. AdMSC viability was comparable between 2 and 3D cultures, indicating that dHAM scaffolds effectively support cell survival regardless of the culture technique. The composition and properties of dHAM preserved cell functions in both culture systems. Flow cytometry confirmed the multilineage differentiation potential of AdMSCs. FESEM imaging revealed AdMSCs with extending filopodia on the scaffold surface and cell penetration up to 17.68µm into the dHAM matrix. The successful 3D recellularization of dHAM with AdMSCs demonstrates its potential as a biological scaffold for stem cell delivery. This approach holds promise for tissue repair and wound healing applications, enhancing the regenerative efficacy of dHAM-based constructs.