ECM Scaffold Research Articles

Biphasic ECM assembled graphene oxide-collagen/nHap composites mimicking articular microenvironment for in situ osteochondral defect repair

Both bone and cartilage microenvironments play a crucial role in the regeneration of osteochondral defects caused by osteoarthritis (OA). A major challenge is how to simulate gradient changing mechanical properties and inducing abilities of regenerative microenvironments on the integrated scaffold to achieve simultaneous regeneration. In this work, a concept of biphasic ECM assembled graphene oxide-collagen (GO-COL)/ Nano-hydroxyapatite (nHap) composite is proposed. GO-COL/nHap bilayer scaffold with gradient concentration was formed by crosslinking and freeze-drying in stages to mimic the gradient distribution of mechanical properties of articular cartilage and bone. Then we prepared chondrogenic/osteogenic ECM membranes (CgECM/OgECM) with chondrocytes from rat knee respectively, and assembled them onto corresponding layers. The morphology, physical properties, biocompatibility, and osteochondrogenic capability of the scaffold were evaluated in vitro and in vivo. Mechanically, gene expression and protein level assessments showed greater osteochondrogenic activity in the biphasic ECM scaffold group compared to the GO-COL bilayer scaffold group. In vivo studies using a rat knee osteochondral defect model further confirmed the superior repair effect of the biphasic ECM-scaffold group compared to the blank group. Taken together, the proposed ECM assembled GO-COL/nHap strategy open a window to realize different microenvironment mimicking in a whole scaffold, making it a potential off-the-shelf method for osteochondral tissue engineering.

Melatonin Promotes Differentiation of Human Spermatogonial Stem Cells Cultured on Three-Dimensional Decellularized Human Testis Matrix.

The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions. SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay. The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively. Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Peran Decellurarized Dental Pulp Sebagai Kandidat Biomaterial Baru Dalam Regenerasi Pulpa : Tinjauan Literatur

Pulp necrosis is one of the main diseases that are most often found in the teeth and mouth. Root canal treatment is the main solution for pulp necrosis, but its implementation can lead to tooth death and recurrent infections. Regenerating the pulp by transplanting scaffolds, stem cells, and growth factors into the root canal is an alternative that can be offered to treat pulpal disease by keeping the teeth vital. Scaffold plays an important role in pulp regeneration, one of which is the ECM scaffold derived from pulp which combines the micro-collagen structure along with bioactive molecules that are important for pulp regeneration and able to promote the differentiation and proliferation of stem cells. This literature review aimed to identify the role of decellularized dental pulp as a new biomaterial candidate in pulp regenerations. The method being used in this review is systematic literature review using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) method. Decellularized Dental Pulp as a New Biomaterial Candidate has a role in pulp regeneration.

Proteome of Personalized Tissue-Engineered Veins.

Vascular diseases are the largest cause of death globally and impose a major global burden on healthcare. The gold standard for treating vascular diseases is the transplantation of autologous veins, if applicable. Alternative treatments still suffer from shortcomings, including low patency, lack of growth potential, the need for repeated intervention, and a substantial risk of developing infections. The use of a vascular ECM scaffold reconditioned with the patient's own cells has shown successful results in preclinical and clinical studies. In this study, we have compared the proteomes of personalized tissue-engineered veins of humans and pigs. By applying tandem mass tag (TMT) labeling LC/MS-MS, we have investigated the proteome of decellularized (DC) veins from humans and pigs and reconditioned (RC) DC veins produced through perfusion with the patient's whole blood in STEEN solution, applying the same technology as used in the preclinical studies. The results revealed high similarity between the proteomes of human and pig DC and RC veins, including the ECM texture after decellularization and reconditioning. In addition, functional enrichment analysis showed similarities in signaling pathways and biological processes involved in the immune system response. Furthermore, the classification of proteins involved in immune response activity that were detected in human and pig RC veins revealed proteins that evoke immunogenic responses, which may lead to graft rejection, thrombosis, and inflammation. However, the results from this study imply the initiation of wound healing rather than an immunogenic response, as both systems share the same processes, and no immunogenic response was reported in the preclinical and clinical studies. Finally, our study assessed the application of STEEN solution in tissue engineering and identified proteins that may be useful for the prediction of successful transplantations.

Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

A scalable corneal xenograft platform: simultaneous opportunities for tissue engineering and circular economic sustainability by repurposing slaughterhouse waste.

Introduction: Corneal disease is a leading cause of blindness globally that stems from various etiologies. High-throughput platforms that can generate substantial quantities of corneal grafts will be invaluable in addressing the existing global demand for keratoplasty. Slaughterhouses generate substantial quantities of underutilized biological waste that can be repurposed to reduce current environmentally unfriendly practices. Such efforts to support sustainability can simultaneously drive the development of bioartificial keratoprostheses. Methods: Scores of discarded eyes from the prominent Arabian sheep breeds in our surrounding region of the United Arab Emirates (UAE) were repurposed to generate native and acellular corneal keratoprostheses. Acellular corneal scaffolds were created using a whole-eye immersion/agitation-based decellularization technique with a widely available, eco-friendly, and inexpensive 4% zwitterionic biosurfactant solution (Ecover, Malle, Belgium). Conventional approaches like DNA quantification, ECM fibril organization, scaffold dimensions, ocular transparency and transmittance, surface tension measurements, and Fourier-transform infrared (FTIR) spectroscopy were used to examine corneal scaffold composition. Results: Using this high-throughput system, we effectively removed over 95% of the native DNA from native corneas while retaining the innate microarchitecture that supported substantial light transmission (over 70%) after reversing opacity, a well-established hallmark of decellularization and long-term native corneal storage, with glycerol. FTIR data revealed the absence of spectral peaks in the frequency range 2849cm-1 to 3075cm-1, indicating the effective removal of the residual biosurfactant post-decellularization. Surface tension studies confirmed the FTIR data by capturing the surfactant's progressive and effectual removal through tension measurements ranging from approximately 35mN/m for the 4% decellularizing agent to 70mN/m for elutes highlighting the effective removal of the detergent. Discussion: To our knowledge, this is the first dataset to be generated outlining a platform that can produce dozens of ovine acellular corneal scaffolds that effectively preserve ocular transparency, transmittance, and ECM components using an eco-friendly surfactant. Analogously, decellularization technologies can support corneal regeneration with attributes comparable to native xenografts. Thus, this study presents a simplified, inexpensive, and scalable high-throughput corneal xenograft platform to support tissue engineering, regenerative medicine, and circular economic sustainability.

Faster and Protective Wound Healing Mechanistic of Para‐Coumaric Acid Loaded Liver ECM Scaffold Cross‐linked with Acellular Marine Kelp (Adv. Funct. Mater. 17/2023)

Natural Crosslinkers In article number 2212325, Byong-Taek Lee and coworkers develop a highly functional wound dressing using acellular marine kelp crosslinked ECM proteins and p-Coumaric acid (p-Cou). Incorporating p-Cou accelerates the healing mechanisms and maintains a unique pH balance of the biomaterial. The study is a noble approach toward the preparation of functional materials and analyzing their functionality by combining molecular simulation.

Hierarchical porous ECM scaffolds incorporating GDF-5 fabricated by cryogenic 3D printing to promote articular cartilage regeneration

BackgroundIn recent years, there has been significant research progress on in situ articular cartilage (AC) tissue engineering with endogenous stem cells, which uses biological materials or bioactive factors to improve the regeneration microenvironment and recruit more endogenous stem cells from the joint cavity to the defect area to promote cartilage regeneration.MethodIn this study, we used ECM alone as a bioink in low-temperature deposition manufacturing (LDM) 3D printing and then successfully fabricated a hierarchical porous ECM scaffold incorporating GDF-5.ResultsComparative in vitro experiments showed that the 7% ECM scaffolds had the best biocompatibility. After the addition of GDF-5 protein, the ECM scaffolds significantly improved bone marrow mesenchymal stem cell (BMSC) migration and chondrogenic differentiation. Most importantly, the in vivo results showed that the ECM/GDF-5 scaffold significantly enhanced in situ cartilage repair.ConclusionIn conclusion, this study reports the construction of a new scaffold based on the concept of in situ regeneration, and we believe that our findings will provide a new treatment strategy for AC defect repair.

Mid- to Long-Term Clinical Outcomes of Cartilage Restoration of Knee Joint with Allogenic Next-Generation Matrix-Induced Autologous Chondrocyte Implantation (MACI).

Cartilage defect is a common pathology still lacking a unified treating option. The purpose of this retrospective study is to evaluate the safety, efficacy, and clinical and radiological outcome of cartilage restoration of knee joint with allogenic next-generation Matrix-Induced Autologous Chondrocyte Implantation (MACI) for the first time, as well as the correlation between postoperative clinical and radiological outcomes and preoperative patient history and demographics. From July 2014 to August 2020, 15 patients who went through cartilage restoration with allogenic next-generation MACI were included in this study. Patient demographics and PROM including the International Knee Documentation Committee (IKDC) subjective knee score, Lysholm score, Tegner Activity Scale (TAS), and Knee Injury and Osteoarthritis Outcome Score (KOOS) were obtained preoperatively, at 3, 6, 12months postoperatively and the last follow-up using an online questionnaire platform. MOCART 2.0 score was calculated at the last follow-up. Analysis of variance (ANOVA) was used to compare PROM pre- and post-operation, with two-tailed p < 0.05 defined as statistical significant. Pearson correlation coefficient was used to evaluate correlation between the PROM and MOCART 2.0 score at the last follow-up with patients demorgraphics. All patients were followed for an average of 66.47± 24.15months (range, 21-93). All patients were satisfied with the outcome of the surgery and no complication was reported at the end of the study. No significant improvement was observed until 1 year after the implantation, except for IKDC score at 6 months. All PROM showed significant improvement 1 year post-op except for Lysholm score and TAS, which also increased significantly at the time of the last follow-up. Pearson correlation coefficient showed that the size of the defect, before or after debridement, was significantly negatively correlated with final KOOS-Pain (before debridement: r=-0.57, p < 0.05; after debridement: r=-0.54, p < 0.05) and KOOS-Symptoms score (before debridement: r=-0.66, p < 0.05; after debridement: r=-0.67, p < 0.05). The MOCART 2.0 score was found significantly and negatively correlated with BMI (r=-0.60, p < 0.05), and significantly and positively correlated with Lysholm score (r=0.70, p < 0.05). The next generation MACI with autologous chondrocyte and allogenic chondrocyte ECM scaffold could be used to treat focal articular cartilage defect in the knee joint safely and efficiently with lasting promising outcomes for more than 5 years. The size of the defects should be considered the most negatively correlated parameters influencing the postoperative clinical outcomes.

Effect of Co-culture of mesenchymal stem cell and glomerulus endothelial cell to promote endothelialization under optimized perfusion flow rate in whole renal ECM scaffold.

In recent era, many researches on implantable bio-artificial organs has been increased owing to large gap between donors and receivers. Comprehensive organ based researches on perfusion culture for cell injury using different flow rate have not been conducted at the cellular level. The present study investigated the co-culture of rat glomerulus endothelial cell (rGEC) and rat bone marrow mesenchymal stem cells (rBMSC) to develop micro vascularization in the kidney scaffolds culturing by bioreactor system. To obtain kidney scaffold, extracted rat kidneys were decellularized by 1% sodium dodecyl sulfate (SDS), 1% triton X-100, and distilled water. Expanded rGECs were injected through decellularized kidney scaffold artery and cultured using bioreactor system. Vascular endothelial cells adhered and proliferated on the renal ECM scaffold in the bioreactor system for 3, 7 and 14 days. Static, 1​ml/min and 2​ml/min flow rates (FR) were tested and among them, 1​ml/min flow rate was selected based on cell viability, glomerulus character, inflammation/endothelialization proteins expression level. However, the flow injury was still existed on primary cell cultured at vessel in kidney scaffold. Therefore, co-culture of rGEC​+​rBMSC found suitable to possibly solve this problem and resulted increased cell proliferation and micro-vascularization in the glomerulus, reducing inflammation and cell death which induced by flow injury. The optimized perfusion rate under rGEC​+​rBMSC co-culture conditions resulted in enhanced endocellularization to make ECM derived implantable renal scaffold and might be useful as a way of treatment of the acute renal failure.

Generating Large Numbers of Pancreatic Microtumors on Alginate-Gelatin Hydrogels for Quantitative Imaging of Tumor Growth and Photodynamic Therapy Optimization.

The emerging use of 3D culture models of cancer has provided novel insights into the therapeutic mechanisms of photodynamic therapy on a mesoscopic scale. Especially microscale tumors grown on scaffolds of extracellular matrix can provide statistically robust data on the effects of photosensitizers and photodynamic therapy by leveraging high-throughput imaging-based assays. Although highly informative, the use of such 3D cultures can be impractical due to the high costs and inter-batch variability of the extracellular matrix scaffolds that are necessary to establish such cultures. In this study, we therefore provide a protocol to generate inexpensive and defined hydrogels composed of sodium alginate and gelatin that can be used for culturing 3D microtumors in a manner that is compatible with state-of-the-art imaging assays. Our results reveal that the alginate-gelatin hydrogels can perform similarly to a commercially available ECM scaffold in terms of facilitating microtumor growth. We then applied these microtumor models to quantify the uptake and dark toxicity of benzoporphyrin derivative encapsulated in liposomes with either an anionic or a cationic surface charge. The results indicate that cationic liposomes achieve the highest level of uptake in the microtumors, yet also exert minor toxicity. Moreover, we reveal that there is typically a significant positive correlation between microtumor size and liposome uptake. In conclusion, alginate-based hydrogels are inexpensive and effective scaffolds for 3D culture models of cancer, with versatile applications in research toward photodynamic therapy.

Decellularized sturgeon cartilage extracellular matrix scaffold inhibits chondrocyte hypertrophy in vitro and in vivo.

Since chondrocyte hypertrophy greatly limits the efficiency of cartilage defects repairing via cartilage tissue engineering (CTE), it is critical to develop a functional CTE scaffold able to inhibit chondrocyte hypertrophy during this period of cartilage regeneration. In this study, we tested the applicability of using decellularized sturgeon cartilage ECM (dSCECM) scaffold to cease chondrocyte hypertrophy during cartilage damage repair. The dSCECM scaffolds with interconnected porous structure and pore size of 114.1±20.9μm were successfully prepared with freeze-dry method. Chondrocytes displayed a round shape and aggregated to form cellular spheroids within dSCECM scaffolds, which is similar to their chondrocytic phenotype within cartilage in vivo. Higher transcriptional level of chondrogenic related genes and integrin related genes was observed in chondrocytes incubated with dSCECM scaffolds instead of type I collagen (COL I) scaffolds, which were used as the control due to their widely usage in CTE and clinic applications. Furthermore, it confirmed that, compared with COL I scaffolds, dSCECM scaffolds significantly reduced the transcription of chondrocyte hypertrophy related genes in chondrocytes following the hypertrophic induction treatment. To test the ability of dSCECM scaffold to inhibit chondrocytes hypertrophy in vivo, chondrocytes with dSCECM scaffolds and COL I scaffolds were cultured with hypertrophic media and were implanted into nude mice respectively. Following 4weeks implantation, interestingly, only the specimens derived from COL I scaffolds displayed consequences of chondrocyte hypertrophy like calcification deposition, demonstrating that chondrocyte hypertrophy is ceased by the dSCECM scaffold following hypertrophic induction. It suggests that the dSCECM scaffold can be potentially applied in clinical treating cartilage defects via the CTE approach to avoid the risk of chondrocyte hypertrophy.

Lung Extracellular Matrix as a Platform for Lung Organ Bioengineering: Design and Development of Tissue Engineered Lung.

Since lung tissue is not able to be reconstructed after substantial injury, lung transplantation often is the only alternative for treatment. Antibiotic-resistant organisms that remain in donor lungs causing infection in the immunosuppressed recipient are among the complications following transplantation. Development of strategies for whole lung regeneration is a pleasing choice particularly in patients with end-stage lung diseases. Reconstruction of lung tissue in vitro for transplantation received increased attention which could deal with the shortage of donor organs. Recent advancements in the field of tissue engineering and regenerative medicine have paved the road for beneficial alternative therapies. Our group has extensive experience with regard to the structure of the lung tissue, which makes us to our decision to continue with the preparation of lung, with the aim of developing a new ECM scaffold. Herein, we aim to review the state-of-art and the tissue engineering and regenerative medicine technology highlighting the major achievements toward the production of a bioengineered lung obtained decellularization and recellularization techniques. We have strong hopes that recent developments in the engineering of lung will lead to similar breakthroughs in the engineering of distal lung components in the future.

Experimental study on the repair of ureteral functional regeneration with highly bioactive extracellular matrix stent.

The research of extracellular matrix stent (ECM) has made some progress in the repair of urethra and bladder defects. To observe the effects of highly bioactive ECM scaffold on the regeneration and repair of defects in long-segment ureteral replacement. An animal model of long-segment ureteral defect was established and four-layer tubular highly bioactive ECM materials were prepared. After the ureteral defect was repaired through surgery, the rabbits in the negative control group were administered a non-bioactive stent, and rabbits in the observation group were treated with an ECM stent. Comparison of macro-indicators: The negative control group had a higher infection rate, a lower survival rate and more complications than the observation group (p < 0.05). The frequency of ureteral peristalsis in the negative control group was lower than in the observation group. In addition, the rate of urinary dysfunction was higher, and the ratio of ureteral diameter was lower in the negative control group than in the observation group (all p < 0.05). Comparison of histopathology: Three months after the operation, the vascular, smooth muscle and mucous membrane of the ureter in the observation group regenerated to close to normal ureteral tissue. There was no significant difference between the ureter regeneration in the repair area and the normal ureter tissue in the observation group 3 months after the operation. The number of regenerated muscle fibers in the observation group was significantly higher than that of the negative control group. Compared with the negative control group, the fibrous capsule was thicker, the percentages of CD31, CD3, CD68, CD80+, and CD163+ were higher, the scope of new smooth muscle fiber was expanded, fusion with the host muscle fibers was higher, and the neuromuscular junction (NMJ) structure was stronger in the observation group (all p < 0.05). A highly bioactive ECM stent can better regenerate the local anatomical structure and physiological function.

Creation of Bony Microenvironment with Extracellular Matrix Doped-Bioactive Ceramics to Enhance Osteoblast Behavior and Delivery of Aspartic Acid-Modified BMP-2 Peptides.

IntroductionDecellularized matrix from porcine small intestinal submucosa (SIS) endows scaffolds with an ECM-like surface, which enhances stem cell self-renewal, proliferation, and differentiation. Mesoporous bioactive glass (MBG) is extensively recognized as an excellent bio-ceramic for fabricating bone grafts.Materials and MethodsIn the current study, SIS was doped on an MBG scaffold (MBG/SIS) using polyurethane foam templating and polydopamine chemistry method. To mimic the bony environment of a natural bone matrix, an ECM-inspired delivery system was constructed by coupling the BMP2-related peptide P28 to a heparinized MBG/SIS scaffold (MBG/SIS-H-P28). The release of P28 from MBG/SIS-H-P28 and its effects on the proliferation, viability, and osteogenic differentiation of bone marrow stromal stem cells were investigated in vitro and in vivo.ResultsOur research indicated that the novel tissue-derived ECM scaffold MBG/SIS has a hierarchical and interconnected porous architecture, and superior biomechanical properties. MBG/SIS-H-P28 released P28 in a controlled manner, with the long-term release time of 40 d. The results of in vitro experiments showed improvements in cell proliferation, cell viability, alkaline phosphatase activity, and mRNA expression levels of osteogenesis-related genes (Runx-2, OCN, OPN, and ALP) compared to those of MBG/SIS or MBG/SIS-P28 and MBG/SIS-H-P28. The in vivo results demonstrated that MBG/SIS-H-P28 scaffolds evidently increased bone formation in rat calvarial critical-sized defect compared to that in controls.ConclusionMBG/SIS-H-P28 scaffolds show potential as ideal platforms for delivery of P28 and for providing a bony environment for bone regeneration.

A cancellous bone matrix system with specific mineralisation degrees for mesenchymal stem cell differentiation and bone regeneration.

Bone regenerative therapies have been explored using various biomaterial systems. Notably, collagen biomineralisation is believed to be essential for promoting bone regeneration. However, ideal bone repair materials with an appropriate mineralised matrix, superior osteogenic activity with early vascularisation, and recellularisation properties are still needed. This study aimed to develop a method to subject the decellularised cancellous bone matrix (DCBM) to ultrasound to obtain specific demineralisation to investigate the effects of DCBM with different degrees of mineralisation on proliferation and osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) and in repairing femoral bone defects in rabbits. We established an optimised native DCBM mineralisation ECM scaffold for bone regeneration. Upon complete decellularisation of the cancellous bone matrix, DCBMs with specific degrees of mineralisation were obtained. We comprehensively evaluated their bioactive components, minimal immunogenicity, ultra-micro-structural mechanical properties, and degree of mineralisation. Furthermore, specific mineralised DCBMs (obtained by low-temperature rapid ultrasound for 4 and 8 h) had prominent effects in promoting the osteogenic differentiation of BMSCs in vitro. Moreover, more newly formed trabeculae, vessels, and endochondral bone were also detected in the aforementioned groups during early-stage bone repair in vivo. The underlying mechanism might be mineralisation-related regulation and ultra-micro-structural mechanical properties. Thus, the present study shows that specific demineralised DCBM obtained under optimal conditions had superior properties to those of unmineralised or completely demineralised DCBM by promoting MSC osteogenic differentiation and initiating endochondral bone formation and de novo osteogenesis.

In vitro artificial skin engineering by decellularized placental scaffold for secondary skin problems of meningomyelocele

BackgroundMeningomyelocele (MMC) is a condition that is originated by the fusion defect of the neural tube. It is a congenital anomaly and can be characterized by spinal cord defects and impaired skin integrity. It is very important to close the skin openings via three-dimensional artificial skin like construction for preventing infection and maintaining the healthy skin structure. Therefore, we aim to generate artificial skin like structures formed by the own cells of donor for treating the MMC-related skin disorder. MethodsIn this study, waste placental tissues were collected and decellularization process was applied. Decellularized and normal placental tissues were compared by immunohistochemistry (IHC). Donor’s own placental stem cells were seeded onto biological scaffold and were differentiated into skin related cell types. Finally, gene expressions were evaluated, and the structural integrity were analyzed with IHC. Tube formation assay was also performed for examining the angiogenesis formation of the tissue in order to evaluate the possibility of a healthy organ development. ResultsCharacterization experiments proved that the decellularized skin preserved a normal skin 3D construction and vasculature along with significant ECM arrangements. The well-kept placental ECM scaffold was cytocompatible, supportive of mesenchymal cell types. Native organ related scaffold is expected to carry a huge influence in skin tissue engineering via delivering a niche for skin-based cells and even for stem/progenitor cells. Regarding to the data obtained from this study, in vivo investigation the skin-like structure in animal models is thought to be the next step as a future prospect. ConclusionThis study is a reference investigation for skin engineering based on placental stem cells and biological scaffolds.

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three-dimensional prevascularized tissues for diverse applications.

Self-sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi-potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three-dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co-culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O2 ) was introduced during the 7day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O2 ) for up to 7days. The results showed that a branched and mature vascular network was formed in the co-culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) were significantly increased by low-oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi-potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three-dimensional prevascularized tissues for diverse applications.

Long‐term development and culture of artificial breast tumor tissue using natural de‐cellularized ECM scaffold materials and the adenocarcinoma cell line MCF‐7

Over the past decade our laboratory, the UWRF Tissue and Cellular Innovation Center has been engaged in a series of studies exploring the application of natural de‐cellularized ECM materials as scaffolds for the generation of 3D artificial tissues. In these studies, we have repeatedly observed the development of significant tissues with extremely long culture lives. In fact, we regularly generate and maintain both tumor and “normal” tissues from standard ATCC cell lines which establish a distinct “tissue homeostasis” leading to long‐term growth and maintenance of complex structures. In recent studies with breast cancer adenocarcinoma cells, MCF‐7 and normal breast ductal cells, MCF10A, viable artificial tissues have been established and maintained for longer than 10 months on a natural marine invertebrate scaffold. Following that time in continuous culture both tumor and normal artificial tissues are still shedding viable cells into their respective wells and generating new monolayers. In both cases these display significant evidence of spontaneous differentiation as well as strong growth characteristics. More significantly, in a related series of studies, MCF‐7‐based artificial tissues have been maintained on de‐cellularized and decalcified bovine trabecular bone scaffolds for longer than 3.5 years of continuous culture. Once again the tissues shed cells into the wells and formed new monolayers continuously throughout the entire culture period. These monolayers consistently displayed a phenotype which includes large numbers of cells generating ridges and potential duct‐like tube structures. Subcultures from regular intervals during this long culture period, 3.5 years, are undergoing further analysis and comparison to explore changes in cell population with an eye to the modeling of tumor progression over the culture period.

Treatment of rabbit growth plate injuries with oriented ECM scaffold and autologous BMSCs

Tissue-engineered technology has provided a promising method for the repair of growth plate injuries using biocompatible and biodegradable scaffolds and appropriate cells. The aim of this study was to fabricate oriented ECM scaffolds to imitate the material and structure of a natural growth plate and to investigate whether BMSCs in a scaffold could prevent the formation of bone bridges in an injured growth plate. We developed a natural, acellular and oriented scaffold derived from a growth plate. The oriented scaffold was fabricated using new freeze-drying technology and by cross-linking the microfilaments in the growth plate. From histological examination, the scaffold contained most of the ECM components including GAG and collagen II without cell DNA fragments, and SEM revealed that oriented scaffold had a uniform aperture in the transverse plane and columnar structure in length plane. Cytotoxicity testing with MTT showed no cytotoxic effect of the scaffold extracts on BMSCs. Autogenous BMSCs in oriented scaffolds promoted the regeneration of neogenetic growth plate when repairing an injured growth plate and prevent the formation of bone bridges to reduce the angular deformity and length discrepancy in the proximal tibia in rabbits. The well-characterized ECM-derived oriented growth plate scaffold shows potential for the repair of injured growth plates in young rabbits.