Autologous cells are usually preferred in treating damaged tissue to avoid risks of immunological rejection and transmitting infectious diseases. Since only limited amount of tissue can be obtained without causing morbidity at the donor site, in vitro expansion of isolated cell is essential in order to acquire sufficient number of cells to reconstruct neocartilage. The aim of this study was to examine whether serial expanded chondrocytes can be use to generate neocartilage in vivo.
A novel decellularization method using sonication treatment is described. Sonication treatment is the combination of physical and chemical agents. These methods will disrupt cell membrane and release cell contents to external environments. The cell removal was facilitated by subsequent rinsing of sodium dodecyl sulfate detergents. Sonication treatment is used in the preparation of complete decellularized bioscaffolds. The aim of this study is to confirm the usefulness of sonication treatment for preparation of biological scaffolds. In this study, samples of aortic tissues are decellularized by sonication treatment at frequency of 170 kHz in 0.1% and 2% sodium dodecyl sulfate detergents for 10-h treatment time. The relation between decellularization and sonication parameters such as dissolved oxygen concentration, conductivity, and pH is investigated. Histological analysis and biomechanical testing is performed to evaluate cell removal efficiency as well as changes in biomechanical properties. Minimal inflammation response elicit by bioscaffolds is confirmed by xenogeneic implantation and immunohistochemistry. Sonication treatment is able to produce complete decellularized tissue suggesting that these treatments could be applied widely as one of the decellularization method.
To avoid tissue rejection during organ transplantation, research has focused on the use of tissue engineering to regenerate required tissues or organs for patients. The biomedical applications of hyperbranched, multivalent, structurally uniform, biocompatible dendrimers in tissue engineering include the mimicking of natural extracellular matrices (ECMs) in the 3D microenvironment. Dendrimers are unimolecular architects that can incorporate a variety of biological and/or chemical substances in a 3D architecture to actively support the scaffold microenvironment during cell growth. Here, we review the use of dendritic delivery systems in tissue engineering. We discuss the available literature, highlighting the 3D architecture and preparation of these nanoscaffolds, and also review challenges to, and advances in, the use dendrimers in tissue engineering. Advances in the manufacturing of dendritic nanoparticles and scaffold architectures have resulted in the successful incorporation of dendritic scaffolds in tissue engineering.
Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid) (PLGA) hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.
Introduction: Oral cancer is the sixth most common malignancy in the world. It is a major concern in Southeast Asia primarily due to betel quid chewing, smoking, and alcohol consumption. In Malaysia, oral cancer related cases accounts for 1.55% of the cause of deaths. Despite recent advances in cancer diagnoses and therapies, the survival rate of oral cancer patients only reached 50% in the last few decades. Tissue engineering (TE) principles may pro-vide new technology platforms to study mechanisms of angiogenesis and tumour cell growth as well as potentially tumour cell spreading in cancer research. The use of biomaterial, appropriate cell source and proper signalling mol-ecules are vital components of TE. Collagen biomaterial are widely used scaffold or membrane in oral application. Nevertheless, no review has been performed on the its usage for the study of oral cancer. This study aimed to sys-tematically review the use of collagen scaffold in oral cancer application. Methods: Research articles were searched using Scopus, Pubmed and Web of Science (WOS) databases. The keywords were limited to “collagen membrane OR collagen scaffold” AND “oral cancer”. Results: Initial search yielded 61 papers (Scopus:37, Pubmed: 12, WOS: 12). Further scrutinization of the papers based on the inclusion criteria resulted total of 3 papers. Two of the papers used collagen membrane for regeneration of oral mucosal defect and increment of alveolar ridge height post-surgery. The remaining paper utilize collagen biomaterial as scaffold for the culture of adenoid cystic carcinoma (ACC) cells. All papers reported significant role of collagen biomaterial in terms of tissue formation, healing scaffold and cellular proliferation. Conclusion: Collagen utilization as biomaterial offers potential use for regeneration of oral related structures as well providing useful model for therapeutics anti-cancer research.
Bone marrow stem cells (BMSC), known for its multipotency to differentiate into various mesenchymal cells such as chodrocyte, osteoblasts, adipocytes, etc, have been actively applied in tissue engineering. BMSC have been successfully isolated from bone marrow aspirate and bone marrow scraping from patients of various ages (13-56 years) with as little as 2ml to 5ml aspirate. BMSC isolated from our laboratory showed the presence of a heterogenous population that showed varying prevalence of surface antigens and the presence of telomerase activity albeit weak. Upon osteogenic induction, alkaline phosphatase activity and mineralization activity were observed.
The purpose of this study was to compare the use of autologous fibrin to human amniotic membrane (HAM) as a scaffold in cultivating autologous conjunctiva for transplantation in treatment of conjunctival defect. An experimental study was performed using 18 adult New Zealand white strain rabbits which were divided into 3 groups. Each group consists of 6 rabbits. The conjunctiva on the temporal site was excised to create a conjunctival epithelial defect. The excised area in the Group 1 was transplanted with autologous conjunctiva cultivated on autologous fibrin; Group 2 was transplanted with autologous conjunctiva cultivated on HAM and Group 3 was left bare. The rabbits were followed up at regular intervals until 6 weeks. The mean period of complete conjunctival epithelization was 11.50 ± 8.22 days for the autologous fibrin group, 15.33 ± 11.80 days for the HAM group and 25.33 ± 5.32 days in the bare sclera group. The epithelization rate for the autologous fibrin group was faster compared to the other two groups. However all the results were not statistically significant (p value >0.05). There were no postoperative complications noted during the follow up. Autologous fibrin is comparable to HAM as a scaffold for cultivation of conjunctiva in the treatment of conjunctival defect.
Animal serum is commonly used in chondrocytes culture expansion to promote cell proliferation and shorten the time lag before new tissue reconstruction is possible. However, animal serum is not suitable for regeneration of clinical tissue because it has potential risk of viral and prion related disease transmission particularly mad cow disease and foreign protein contamination that can stimulate immune reaction leading to graft rejection. In this context, human serum as homologous supplement has a greater potential as growth promoting agents for human chondrocytes culture.
Cellulose is a natural homopolymer, composed of β-1,4- anhydro-d-glucopyranose units. Unlike plant cellulose, bacterial cellulose (BC), obtained from species belonging to the genera of Acetobacter, Rhizobium, Agrobacterium, and Sarcina through various cultivation methods and techniques, is produced in its pure form. BC is produced in the form of gel-like, never dry sheet with tremendous mechanical properties. Containing up to 99% of water, BC hydrogel is considered biocompatible thus finding robust applications in the health industry. Moreover, BC three-dimensional structure closely resembles the extracellular matrix (ECM) of living tissue. In this review, we focus on the porous BC morphology particularly suited to host oxygen and nutrients thus providing conducive environment for cell growth and proliferation. The remarkable BC porous morphology makes this biological material a promising templet for the generation of 3D tissue culture and possibly for tissue-engineered scaffolds.
Articular cartilage is well known for its simple uniqueness of avascular and aneural structure that has limited capacity to heal itself when injured. The use of three dimensional construct in tissue engineering holds great potential in regenerating cartilage defects. This study evaluated the in vitro cartilaginous tissue formation using rabbit's bone marrow mesenchymal stem cells (BMSCs)-seeded onto poly(lactic-co-glycolic acid) PLGA/fibrin and PLGA scaffolds. The in vitro cartilaginous engineered constructs were evaluated by gross inspection, histology, cell proliferation, gene expression and sulphated glycosaminoglycan (sGAG) production at week 1, 2 and 3. After 3 weeks of culture, the PLGA/fibrin construct demonstrated gross features similar to the native tissue with smooth, firm and glistening appearance, superior histoarchitectural and better cartilaginous extracellular matrix compound in concert with the positive glycosaminoglycan accumulation on Alcian blue. Significantly higher cell proliferation in PLGA/fibrin construct was noted at day-7, day-14 and day-21 (p<0.05 respectively). Both constructs expressed the accumulation of collagen type II, collagen type IX, aggrecan and sox9, showed down-regulation of collagen type I as well as produced relative sGAG content with PLGA/fibrin construct exhibited better gene expression in all profiles and showed significantly higher relative sGAG content at each time point (p<0.05). This study suggested that with optimum in vitro manipulation, PLGA/fibrin when seeded with pluripotent non-committed BMSCs has the capability to differentiate into chondrogenic lineage and may serve as a prospective construct to be developed as functional tissue engineered cartilage.
Polyhydroxyalkanoates (PHAs) have attracted the attention of academia and industry because of their plastic-like properties and biodegradability. However, practical applications as a commodity material have not materialized because of their high production cost and unsatisfactory mechanical properties. PHAs are also believed to have high-value applications as an absorbable biomaterial for tissue engineering and drug-delivery devices because of their biocompatibility. However, research in these areas is still in its very early stages. The main problem faced by proponents of PHAs is the lack of a niche area where PHAs will be the most desired material in terms of its function during use rather than because of its eco-friendly virtues after use. Here, we report on the oil-absorbing property of PHA films and its potential applications. By comparing with some of the existing commercial products, the potential application of PHAs as cosmetic oil-blotting films is revealed for the first time. Besides having the ability to rapidly absorb and retain oil, PHA films also have a natural oil-indicator property, showing obvious changes in opacity following oil absorption. Surface analysis revealed that the surface structures such as porosity and smoothness exert great influence on the rapid oil-absorption properties of the PHA films. These newly discovered properties could be exploited to create a niche area for the practical applications of PHAs.
This study was performed to determine the microscopic biological response of human nasal septum chondrocytes and human knee articular chondrocytes placed on a demineralized bovine bone scaffold. Both chondrocytes were cultured and seeded onto the bovine bone scaffold with seeding density of 1 x 105 cells per 100 microl/scaffold and incubated for 1, 2, 5 and 7 days. Proliferation and viability of the cells were measured by mitochondrial dehydrogenase activity (MTT assay), adhesion study was analyzed by scanning electron microscopy and differentiation study was analyzed by immunofluorescence staining and confocal laser scanning electron microscopy. The results showed good proliferation and viability of both chondrocytes on the scaffolds from day 1 to day 7. Both chondrocytes increased in number with time and readily grew on the surface and into the open pores of the scaffold. Immunofluorescence staining demonstrated collagen type II on the scaffolds for both chondrocytes. The results showed good cells proliferation, attachment and maturity of the chondrocytes on the demineralized bovine bone scaffold. The bovine bone being easily resourced, relatively inexpensive and non toxic has good potential for use as a three dimensional construct in cartilage tissue engineering.
The actual in vivo tissue scaffold offers a three-dimensional (3D) structural support along with a nano-textured surfaces consist of a fibrous network in order to deliver cell adhesion and signaling. A scaffold is required, until the tissue is entirely regenerated or restored, to act as a temporary ingrowth template for cell proliferation and extracellular matrix (ECM) deposition. This review depicts some of the most significant three dimensional structure materials used as scaffolds in various tissue engineering application fields currently being employed to mimic in vivo features. Accordingly, some of the researchers' attempts have envisioned utilizing graphene for the fabrication of porous and flexible 3D scaffolds. The main focus of this paper is to evaluate the topographical and topological optimization of scaffolds for tissue engineering applications in order to improve scaffolds' mechanical performances.
The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.
This study aimed to isolate, culture-expand and characterize the chondrocytes isolated from microtic cartilage and evaluate its potential as a cell source for ear cartilage reconstruction. Specific attention was to construct the auricular cartilage tissue by using fibrin as scaffold.
In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell's chondrogenic induction.
Culture media supplemented with animal serum e.g. fetal bovine serum; FBS is commonly used for human culture expansion. However, for clinical application, FBS is restricted as its carry a risk of viral or prion transmission. Engineering autologous cartilage with autologous human serum supplementation is seen as a better solution to reduce the risk of transmitting infectious diseases and immune rejection during cartilage transplantation. The purpose of this study is to establish and compare the effects of 10% autologous human serum (AHS) and 10% FBS on the growth of chondrocytes and the formation of tissue engineered human articular cartilage.
Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
For dental implants, it is vital that an initial soft tissue seal is achieved as this helps to stabilize and preserve the peri-implant tissues during the restorative stages following placement. The study of the implant-soft tissue interface is usually undertaken in animal models. We have developed an in vitro three-dimensional tissue-engineered oral mucosal model (3D OMM), which lends itself to the study of the implant-soft tissue interface as it has been shown that cells from the three-dimensional OMM attach onto titanium (Ti) surfaces forming a biological seal (BS). This study compares the quality of the BS achieved using the three-dimensional OMM for four types of Ti surfaces: polished, machined, sandblasted and anodized (TiUnite). The BS was evaluated quantitatively by permeability and cell attachment tests. Tritiated water (HTO) was used as the tracing agent for the permeability test. At the end of the permeability test, the Ti discs were removed from the three-dimensional OMM and an Alamar Blue assay was used for the measurement of residual cells attached to the Ti discs. The penetration of the HTO through the BS for the four types of Ti surfaces was not significantly different, and there was no significant difference in the viability of residual cells that attached to the Ti surfaces. The BS of the tissue-engineered oral mucosa around the four types of Ti surface topographies was not significantly different.
Understanding the bioelectrical properties of bone tissue is key to developing new treatment strategies for bone diseases and injuries, as well as improving the design and fabrication of scaffold implants for bone tissue engineering. The bioelectrical properties of bone tissue can be attributed to the interaction of its various cell lineages (osteocyte, osteoblast and osteoclast) with the surrounding extracellular matrix, in the presence of various biomechanical stimuli arising from routine physical activities; and is best described as a combination and overlap of dielectric, piezoelectric, pyroelectric and ferroelectric properties, together with streaming potential and electro-osmosis. There is close interdependence and interaction of the various electroactive and electrosensitive components of bone tissue, including cell membrane potential, voltage-gated ion channels, intracellular signaling pathways, and cell surface receptors, together with various matrix components such as collagen, hydroxyapatite, proteoglycans and glycosaminoglycans. It is the remarkably complex web of interactive cross-talk between the organic and non-organic components of bone that define its electrophysiological properties, which in turn exerts a profound influence on its metabolism, homeostasis and regeneration in health and disease. This has spurred increasing interest in application of electroactive scaffolds in bone tissue engineering, to recapitulate the natural electrophysiological microenvironment of healthy bone tissue to facilitate bone defect repair.