Tissue engineering scaffolds with oxygen generating elements have shown to be able to increase the level of oxygen and cell survivability in specific conditions. In this study, biphasic calcium phosphate (BCP) scaffolds with the composition of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (β-TCP), which have shown a great potential for bone tissue engineering applications, were fabricated by a direct-write assembly (robocasting) technique. Then, the three-dimensional (3D)-printed scaffolds were coated with different ratios of an oxygen releasing agent, calcium peroxide (CPO), which encapsulated within a polycaprolactone (PCL) matrix through dip-coating, and used for in situ production of oxygen in the implanted sites. The structure, composition and morphology of the prepared scaffolds were characterized by different techniques. The oxygen release kinetics and biological investigations of the scaffolds were also studied in vitro. The results showed that oxygen release behaviour was sustained and dependant on the concentration of CPO encapsulated in the PCL coating matrix. It was also demonstrated that the coated scaffolds, having 3% CPO in the coating system, could provide a great potential for promoting bone ingrowth with improving osteoblast cells viability and proliferation under hypoxic conditions. The findings indicated that the prepared scaffolds could play a significant role in engineering of large bone tissue implants with limitations in oxygen diffusion.
In this study, single, mix, multilayer Polyvinyl alcohol (PVA) electrospun nanofibers with epidermal growth factor (EGF) and fibroblast growth factor (FGF) were fabricated and characterized as a biological wound dressing scaffolds. The biological activities of the synthesized scaffolds have been verified by in vitro and in vivo studies. The chemical composition finding showed that the identified functional units within the produced nanofibers (O-H and N-H bonds) are attributed to both growth factors (GFs) in the PVA nanofiber membranes. Electrospun nanofibers' morphological features showed long protrusion and smooth morphology without beads and sprayed with an average range of 198-286 nm fiber diameter. The fiber diameters decrement and the improvement in wettability and surface roughness were recorded after GFs incorporated within the PVA Nanofibers, which indicated potential good adoption as biological dressing scaffolds due to the identified mechanical properties (Young's modulus) in between 18 and 20 MPa. The MTT assay indicated that the growth factor release from the PVA nanofibers has stimulated cell proliferation and promoted cell viability. In the cell attachment study, the GFs incorporated PVA nanofibers stimulated cell proliferation and adhered better than the PVA control sample and presented no cytotoxic effect. The in vivo studies showed that compared to the control and single PVA-GFs nanofiber, the mix and multilayer scaffolds gave a much more wound reduction at day 7 with better wound repair at day 14-21, which indicated to enhancing tissue regeneration, thus, could be a projected as a suitable burn wound dressing scaffold.
Developing multifaceted, biocompatible, artificial implants for tissue engineering is a growing field of research. In recent times, several works have been reported about the utilization of biomolecules in combination with synthetic materials to achieve this process. Accordingly, in this study, the ability of an extract obtained from Aloe vera, a commonly used medicinal plant in influencing the biocompatibility of artificial material, is scrutinized using metallocene polyethylene (mPE). The process of coating dense fibrous Aloe vera extract on the surface of mPE was carried out using microwaves. Then, several physicochemical and blood compatibility characterization experiments were performed to disclose the effects of corresponding surface modification. The Fourier transform infrared spectrum showed characteristic vibrations of several active constituents available in Aloe vera and exhibited peak shifts at far infrared regions due to aloe-based mineral deposition. Meanwhile, the contact angle analysis demonstrated a drastic increase in wettability of coated samples, which confirmed the presence of active components on glazed mPE surface. Moreover, the bio-mimic structure of Aloe vera fibers and the influence of microwaves in enhancing the coating characteristics were also meticulously displayed through scanning electron microscopy micrographs and Hirox 3D images. The existence of nanoscale roughness was interpreted through high-resolution profiles obtained from atomic force microscopy. And the extent of variations in irregularities was delineated by measuring average roughness. Aloe vera-induced enrichment in the hemocompatible properties of mPE was established by carrying out in vitro tests such as activated partial thromboplastin time, prothrombin time, platelet adhesion, and hemolysis assay. In conclusion, the Aloe vera-glazed mPE substrate was inferred to attain desirable properties required for multifaceted biomedical implants.
Various materials have been used as scaffolds to suit different demands in tissue engineering. One of the most important criteria is that the scaffold must be biocompatible. This study was carried out to investigate the potential of HA or TCP/HA scaffold seeded with osteogenic induced sheep marrow cells (SMCs) for bone tissue engineering.
Calcium silicate (CaSiO3, CS) ceramics are promising bioactive materials for bone tissue engineering, particularly for bone repair. However, the low toughness of CS limits its application in load-bearing conditions. Recent findings indicating the promising biocompatibility of graphene imply that graphene can be used as an additive to improve the mechanical properties of composites. Here, we report a simple method for the synthesis of calcium silicate/reduced graphene oxide (CS/rGO) composites using a hydrothermal approach followed by hot isostatic pressing (HIP). Adding rGO to pure CS increased the hardness of the material by ∼40%, the elastic modulus by ∼52%, and the fracture toughness by ∼123%. Different toughening mechanisms were observed including crack bridging, crack branching, crack deflection, and rGO pull-out, thus increasing the resistance to crack propagation and leading to a considerable improvement in the fracture toughness of the composites. The formation of bone-like apatite on a range of CS/rGO composites with rGO weight percentages ranging from 0 to 1.5 has been investigated in simulated body fluid (SBF). The presence of a bone-like apatite layer on the composite surface after soaking in SBF was demonstrated by X-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM). The biocompatibility of the CS/rGO composites was characterized using methyl thiazole tetrazolium (MTT) assays in vitro. The cell adhesion results showed that human osteoblast cells (hFOB) can adhere to and develop on the CS/rGO composites. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of cells on the CS/rGO composites were improved compared with the pure CS ceramics. These results suggest that calcium silicate/reduced graphene oxide composites are promising materials for biomedical applications.
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.
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.
Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(epsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.
In this study, porous hydroxyapatite (HA) samples were fabricated via sponge techniques with the aid of sago as part of the binder mixture. Development processes for the production of porous bone graft substitutes are studied using polyurethane sponge. To obtain the optimum amount of binder for successful fabrication of porous HA were done. Initially, porous HA powder was synthesized using calcium hydroxide and orthorphosphoric acid. Meanwhile, sago was mixed with PVA in a certain ratio to be used as binder for preparing the porous HA. After a series of investigative tests were conducted to characterize the sintered samples, the use of the sago and polymeric mixture was found to successfully aid the fabrication of porous HA samples. In this investigation, comparison of physical and mechanical characteristics between samples prepared using difference techniques was made.
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.
To evaluate the effect of autologous human serum (AHS) versus pooled human serum (PHS) versus foetal bovine serum (FBS) for growth of articular chondrocytes and formation of chondrocytefibrin constructs.
Normal tracheal mucociliary clearance is the key to maintaining the health and defense of respiratory airway. Therefore the present of cilia and mucous blanket are important for tracheal epithelium to function effectively. In the present study, we prepared a tissue engineered respiratory epithelium construct (TEREC) made of autologous respiratory epithelium cells, fibroblast and fibrin from sheep owns blood which replaced a created tracheal mucosal defect. Scanning electron microscopy (SEM) showed encouraging result where immature cilia were present on the surface of TEREC. This result indicates that engineered respiratory epithelium was able to function as normal tissue.
Bone marrow derived Mesenchymal stem cells (MSCs) were evaluated as an alternative source for tissue engineering of peripheral nerves. Human MSCs were subjected to a series of treatment with a reducing agent, retinoic acid and a combination of trophic factors. This treated MSCs differentiated into Schwann cells were characterized in vitro via flow cytometry analysis and immunocytochemically. In contrast to untreated MSCs, differentiated MSCs expressed Schwann cell markers in vitro, as we confirmed by flow cytometry analysis and immunocytochemically. These results suggest that human MSCs can be induced to be a substitute for Schwann cells that may be applied for nerve regeneration since it is difficult to grow Schwann cells in vitro.
The treatment of major burn injuries are a formidable challenge to the burn surgeon. Early aggressive surgery for deep to full thickness burn injuries is vital in the prevention of infection. The ultimate goal in major burn injuries is to prevent the onset of multi-resistant organisms and achieve early wound cover. The field of tissue engineering can help to expedite the healing of these burn wounds. The development of keratinocyte culture delivery system can be used clinically to fasten the healing process and save many lives.
Tricalcium phosphate/hydroxyapatite (TCP/HA), hydroxyapatite (HA), chitosan and calcium sulphate (CaSO4) were studied and evaluated for possible bone tissue engineered construct acting as good support for osteogenic cells to proliferate, differentiate, and eventually spread and integrate into the scaffold. Surface morphology visualized by SEM showed that scaffold materials with additional fibrin had more cell densities attached than those without, depicting that the presence of fibrin and collagen fibers were truly a favourite choice of cells to attach. In comparison of various biomaterials used incorporated with fibrin, TCP/HA had the most cluster of cells attached.
Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be selectively isolated and induced to express bone phenotype in vitro. The osteoblastic progenitor can be estimated by counting the number of cells attach using the haemacytometer. This study was undertaken to test the hypothesis that human aging is associated with a significant change on the number of osteoblastic progenitors in the bone marrow. Bone marrow aspirates were harvested from 38 patients, 14 men (age 11-70) and 24 women (age 10-70) and cultured in F12: DMEM (1:1). In total 15 bone marrow samples have been isolated from patients above 40 years old (men/women) of age. Fourteen (93.3%) of this samples failed to proliferate. Only one (6.7%) bone marrow sample from a male patient, aged 59 years old was successfully cultured. Seventy percent (16/23) of the samples from patient below than 40 years old were successfully cultured. However, our observation on the survival rate for cells of different gender from patient below 40 years old does not indicate any significant difference. From this study, we conclude that the growth of bone marrow stromal cells possibly for bone engineering is better from bone marrow aspirates of younger patient.
In this study the surface properties of two particulate coral and polyhydroxybutrate (PHB) were studied in order to characterize them prior to use in composite production. Coral powder and PHB particle were evaluated using scanning electron microscopy and confocal laser scanning microscopy, to measure surface porosity and pores size. The results showed that coral powder has multiple pleomorphic micropores cross each others give appearance of micro-interconnectivity. Some pore reached to 18 microm with an average porosity of 70%. PHB revealed multiple different size pores extended to the depth, with an average some times reach 25 microm and porosity 45%. These findings demonstrate that both coral and PHB have excellent pores size and porosity that facilitate bone in growth, vascular invasion and bone development. We believe that incorporation of coral powder into PHB will make an excellent composite scaffold for tissue engineering.
This study was designed to evaluate the ability of natural coral implant to provide an environment for marrow cells to differentiate into osteoblasts and function suitable for mineralized tissue formation. DNA content, alkaline phosptatase (ALP) activity, calcium (Ca) content and mineralized nodules, were measured at day 3, day 7 and day 14, in rat bone marrow stromal cells cultured with coral discs glass discs, while cells alone and coral disc alone were cultured as control. DNA content, ALP activity, Ca content measurements showed no difference between coral, glass and cells groups at 3 day which were higher than control (coral disc alone), but there were higher measurement at day 7 and 14 in the cell cultured on coral than on glass discs, control cells and control coral discs. Mineralized nodules formation (both in area and number) was more predominant on the coral surface than in control groups. These results showed that natural coral implant provided excellent and favorable situation for marrow cell to differentiate to osteoblasts, lead to large amount of mineralized tissue formation on coral surface. This in vitro result could explain the rapid bone bonding of coral in vivo.
Patient own fibrin may act as the safest, cheapest and immediate available biodegradable scaffold material in clinical 1 tissue engineering. This study investigated the feasibility of using patient own fibrin isolated from whole blood to construct a new human cartilage, skin and bone. Constructed in vitro tissues were implanted on the dorsal part of the nude mice for in vivo maturation. After 8 weeks of implantation, the engineered tissues were removed for histological analysis. Our results demonstrated autologous fibrin has great potential as clinical scaffold material to construct various human tissues.