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PubMed2004

Diffusion in musculoskeletal tissue engineering scaffolds: design issues related to porosity, permeability, architecture, and nutrient mixing.

The field of tissue engineering continues to advance with the discovery of new biomaterials, growth factors and scaffold fabrication techniques. However, for the ultimate success of a tissue engineered construct the issue of nutrient transport to the scaffold interior needs to be addressed. Often, the requirements for adequate nutrient supply are at odds with other scaffold design parameters such as mechanical properties as well as scaffold fabrication techniques, leading to incongruities in finding optimal solutions. The goal of this review article is to provide an overview of the various engineering design factors that promote movement of nutrients, waste and other biomolecules in scaffolds for musculoskeletal tissue engineering applications. The importance of diffusion in scaffolds and how it is influenced by porosity, permeability, architecture, and nutrient mixing has been emphasized. Methods for measuring porosity and permeability have also been outlined. The different types of biomaterials used, scaffold fabrication techniques implemented and the pore sizes/porosities obtained over the past 5 years have also been addressed.

Animals↗

PubMed2005

[A new loading bioreactor for bone tissue-engineering applications].

Bone cells live in an environment heavily influenced by mechanical force. The development of bone tissue is dependent on the environment that surrounds it, both in vivo and in vitro. A loading stimulator on research of bone tissue-engineering was developed based on the mechanism of mechanosensation, scaffolding composites with mechanical strains with more physiologic magnitude, frequency components, and waveform. It also achieves the mechanical environment particularly in hard scaffold enough strong like cancellous bone. The device was tested using a reference scaffold made of better elastic plastic material. The experiment results showed that the device could be used in precision strain controls. Since the drive of the stimulator comes from the usage of smart material, piezoceramics, the strain at physiological level is controlled precisely. The stimulator provides a mechanical condition under which the effects of loading applied on bone tissue-engineering culture are conveniently investigated. Furthermore, after the stimulator is improved, it will be an appropriate bioreactor for bone tissue-engineering culture.

Bioreactors↗

PubMed2004

In vivo tissue-engineered small-caliber arterial graft prosthesis consisting of autologous tissue (biotube).

In this study, vascular-like tubular tissues called biotubes, consisting of autologous tissues, were prepared using in vivo tissue engineering. Their mechanical properties were evaluated for application as a small-caliber artificial vascular prosthesis. The biotubes were prepared by embedding six kinds of polymeric rods [poly(ethylene) (PE), poly(fluoroacetate) (PFA), poly(methyl methacrylate) (PMMA), segmented poly(urethane) (PU), poly(vinyl chloride) (PVC), and silicone (Si)] as a mold in six subcutaneous pouches in the dorsal skin of New Zealand White rabbits. For rods apart from PFA, biotubes were constructed after 1 month of implantation by encapsulation around the polymeric implants. The wall thickness of the biotubes ranged from about 50 to 200 microm depending on the implant material and were in the order PFA < PVC < PMMA < PU < PE. As for PE, PMMA, and PVC, the thickness increased after 3 months of implantation and ranged from 1.5-to 2-fold. None of the biotubes were ruptured when a hydrostatic pressure was gradually applied to their lumen up to 200 mmHg. The relationship between the intraluminal pressure and the external diameter, which was highly reproducible, showed a "J"-shaped curve similar to the native artery. The tissue mostly consisted of collagen-rich extracellular matrices and fibroblasts. Generally, the tissue was relatively firm and inelastic for Si and soft for PMMA. For PMMA, PE, and PVC the stiffness parameter (beta value; one of the indexes for compliance) of the biotubes obtained was similar to those of the human coronary, femoral, and carotid arteries, respectively. Biotubes, which possess the ability for wide adjustments in their matrices, mechanics, shape, and luminal surface design, can be applied for use as small-caliber blood vessels and are an ideal implant because they avoid immunological rejection.

Animals↗

PubMed1990

[The determination of trace amounts of protein in solutions containing surface-active substances].

A rapid and sensitive method for protein determination (0.5-16 micrograms) in samples of any volume containing various surfactants in concentration up to 1% is suggested. The method includes the protein acid denaturation, the solution of acid insoluble precipitate of detergent in ethanol (25-30%), the protein determination on nitrocellulose filter, dyeing by aminoblack 10 B, elution of dyed complex and colorimetric determination at 630 nm.

Amido Black↗

PubMed2001

Functional tissue engineering: the role of biomechanics in articular cartilage repair.

Articular cartilage shows little or no intrinsic capacity for repair in response to injury or disease, and even minor lesions or injuries may lead to progressive damage and joint degeneration. Tissue engineering is a relatively new but rapidly growing field that has sought to use combinations of implanted cells, biomaterials, and biologically active molecules to repair or regenerate injured or diseased tissues. Despite many advances, tissue engineers have faced significant challenges in repairing or replacing tissues that serve a predominantly biomechanical function, such as articular cartilage. An evolving discipline termed functional tissue engineering seeks to address these challenges by emphasizing and evaluating the role of biomechanical factors in the intrinsic and engineered repair of tissues and organs. In the current study, the authors describe some of the fundamental issues involving the interaction of biomechanical stresses in vivo and in vitro with native and repair articular cartilage and with other biomechanically functional tissues. A more thorough and formal investigation of these issues may provide a basis for developing rational design principles for tissue engineered replacement or repair of load-bearing structures in the body.

Animals↗

PubMed2001

[The primary study on rabbit bone marrow mesenchymal stem cells in constructing tissue-engineered cartilage].

OBJECTIVE: To study the feasibility of constructing tissue engineered cartilage by differentiated rabbit bone marrow mesenchymal stem cells(MSC) cultured in vitro and in vivo. METHODS: The MSC were isolated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge, and then induced into chondrogenic differentiation by adding dexamethasone, transforming growth factor-beta 1 (TGF-beta 1) and ascorbic acid in vitro. After 3 weeks, some cells turned to round shape and secreted metachromatic matrix. The cartilaginoid grafts composed of chondrogenic MSC. Bovine type I collagen and human fibrin were cultured within the chondrogenic medium for 2 weeks in vitro or transplanted subcutaneously adjacent to the knee joint for 3 weeks in vivo. RESULTS: The most cells in the grafts were degenerated and disappeared after cultured in vitro. But the residual cells were survival and secreted metachromatic staining proteoglycan with toluidine blue, which was characteristic cartilage matrix. The grafts developed into matured cartilage tissue assessed by histological examination after 3 weeks of transplantation in vivo. CONCLUSION: MSC can be used as functional cells to constructing tissue engineered cartilage.

Animals↗

PubMed2006

Techniques for biological characterization of tissue-engineered tendon and ligament.

Injuries to tendons and ligaments are prevalent and result in a significant decrease in quality of patient life. Tissue-engineering strategies hold promise as alternatives to current treatments for these injuries, which often fail to fully restore proper joint biomechanics and produce significant donor site morbidity. Commonly, tissue engineering involves the use of a three-dimensional scaffold seeded with cells that can be directed to form tendon/ligament tissue. When determining the success of such approaches, the viability and proliferation of the cells in the construct, as well as extracellular matrix production and structure should be taken into account. Histology and histochemistry, microscopy, colorimetric assays, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) are techniques that are employed to assess these biological characteristics. This review provides an overview of each of these methods, including specific examples of how they have been used in evaluation of tissue-engineered tendon and ligament tissue. Basic physical principles underlying each method and advantages and disadvantages of the various techniques are summarized.

Animals↗

PubMed2005

Advancing cartilage tissue engineering: the application of stem cell technology.

The treatment of cartilage pathology and trauma face the challenges of poor regenerative potential and inferior repair. Nevertheless, recent advances in tissue engineering indicate that adult stem cells could provide a source of chondrocytes for tissue engineering that the isolation of mature chondrocytes has failed to achieve. Various adjuncts to their propagation and differentiation have been explored, such as biomaterials, bioreactors and growth hormones. To date, all tissue engineered cartilage has been significantly mechanically inferior to its natural counterparts and further problems in vivo relate to poor integration and deterioration of tissue quality over time. However, adult stem cells--with their high rate of proliferation and ease of isolation--are expected to greatly further the development and usefulness of tissue engineered cartilage.

Adipose Tissue↗

PubMed2006

[Structure and performance of calcium polyphosphate for bone tissue engineering].

Porous calcium polyphosphate (CPP) has shown promise of tissue engineered implant application because of the biocompatibility and biodegradation. CPP with different polymerization degree were prepared by controlling the calcining time, and its polymerization degree could be calculated by developed method in this paper. Different crystal types CPP were prepared by quenching from the melt and crystallization of amorphous CPP. From the in vitro degradation, carried out in Tris-HCl buffer, the degradation velocity of CPP was controllable. The weight loss of CPP with different polymerization degrees and crystal types were different. With the increasing of polymerization degree, the weight loss during the degradation was decreasing, contrarily the strength of CPP was increasing. The amorphous CPP could degrade completely in 17 days while gamma-CPP do completely in 25 days. The degradation velocity beta-CPP and alpha-CPP was slower than gamma-CPP and the weight loss was about 12% and 5% respectively. The results of this study indicate that CPP have potential applications for bone tissue engineering as inorganic polymeric biomaterials.

Absorbable Implants↗

PubMed2004

Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities.

An introductory overview of the use of microfluidic devices for tissue engineering is presented. After a brief description of the background of tissue engineering, different application areas of microfluidic devices are examined. Among these are methods for patterning cells, topographical control over cells and tissues, and bioreactors. Examples where microfluidic devices have been employed are presented such as basal lamina, vascular tissue, liver, bone, cartilage and neurons. It is concluded that until today, microfluidic devices have not been used extensively in tissue engineering. Major contributions are expected in two areas. The first is growth of complex tissue, where microfluidic structures ensure a steady blood supply, thereby circumventing the well-known problem of providing larger tissue structures with a continuous flow of oxygen and nutrition, and withdrawal of waste products. The second, and probably more important function of microfluidics, combined with micro/nanotechnology, lies in the development of in vitro physiological systems for studying fundamental biological phenomena.

Animals↗

PubMed1999

The use of tissue-engineered skin (Apligraf) to treat a newborn with epidermolysis bullosa.

BACKGROUND: Inherited epidermolysis bullosa (EB) is a mechanobullous disorder. The Dowling-Meara variant, a subtype of EB, is characterized by widespread blister formation that may include the oral cavity and nails. Many patients with the Dowling-Meara phenotype are at increased risk of sepsis and death during infancy. The treatment of EB is generally supportive. The tissue-engineered skin used (Apligraf) is a bilayered human skin equivalent developed from foreskin. It is the only Food and Drug Administration-approved skin equivalent of its kind. It is approved for the treatment of venous ulcers of the lower extremities. It has also been used to treat acute wounds, such as graft donor sites and cancer excision sites. OBSERVATION: To our knowledge, we describe the first case in which a newborn with EB, Dowling-Meara variant, was treated with bilayered tissue-engineered skin. The areas treated with the tissue-engineered skin healed faster than the areas treated with conventional therapy. Most of the areas treated with tissue-engineered skin have remained healed, without developing new blisters. These areas appear to be more resistant to trauma. CONCLUSIONS: Our early success with tissue-engineered skin in this patient may have a significant impact on the future treatment of neonates with EB simplex. Future studies are needed to determine if the beneficial effects of tissue-engineered skin are reproducible in other neonates with EB simplex and in patients of all ages with different subtypes of EB.

Collagen↗

PubMed2006

A sandwich tubular scaffold derived from chitosan for blood vessel tissue engineering.

Many materials have been investigated in blood vessel tissue engineering, such as PGA, PLGA, P4HB. However, chitosan is not mentioned in the arena. This study aimed to develop a chitosan-based tubular scaffold and examine its feasibility of being applied in this field. Briefly, a knitted chitosan tube was dipped into chitosan solution (2%, w/v) and dried, then its inner and outer surface was mantled with a layer of chitosan/gelatin (4:1, w/w) complex solution, and then freeze-dehydrated. In vitro characterization showed that the scaffold had a wall of 1.0 mm in thickness with a sandwich structure, and a porosity of 81.2%. The pore diameter was 50-150 microm and could be regulated by varying freezing conditions. The scaffold possessed proper swelling property, burst strength of almost 4000 mmHg, and high suture-retention strength. After degradation for 2 months, the scaffold could maintain enough mechanical strength with an average mass loss of 18.7%. Vascular smooth muscle cells could spread and grow very well on the scaffold. This study provided a novel method to fabricate chitosan and its complex into a tubular scaffold and demonstrated the feasibility of the scaffold employed in the field of blood vessel tissue engineering.

Animals↗

PubMed2006

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures.

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.

Aggrecans↗

PubMed2003

A biological hybrid model for collagen-based tissue engineered vascular constructs.

Various approaches to tissue engineering a small diameter blood vessel have historically relied upon extended culturing periods and/or synthetic materials to create mechanical properties suitable to withstand the hemodynamic stresses of the vasculature. In this work, we present the concept of a construct-sleeve hybrid (CSH) graft, which uses a biological support to provide temporary reinforcement while cell-mediated remodeling of the construct occurs. Support sleeves were fabricated from Type I collagen gels and crosslinked with glutaraldehyde, ultraviolet, or dehydrothermal treatments. Uniaxial tensile testing of acellular sleeves revealed increased stiffness moduli and tensile stresses with crosslinking treatments. A second collagen layer containing cells was molded about the sleeve to create a CSH. After in vitro culture, CHSs with uncrosslinked (UnXL) and glutaraldehyde treated (Glut) sleeves exhibited significant increases in mechanical strength (20.4-fold and 121-fold increases in ultimate stress, respectively) compared to unreinforced control constructs. Burst testing produced similar findings with peak pressures of 100 and 650mmHg in the UnXL and Glut CSHs, respectively. Construct compaction, cell viability, and histological examination demonstrated that the function of most cells remained unimpaired with the incorporation of the biological support sleeve.

Biomedical Engineering↗

PubMed2006

Micro-CT-based screening of biomechanical and structural properties of bone tissue engineering scaffolds.

The development of successful scaffolds for bone tissue engineering requires a concurrent engineering approach that combines different research fields. In order to limit in vivo experiments and reduce trial and error research, a scaffold screening technique has been developed. In this protocol seven structural and three biomechanical properties of potential scaffold materials are quantified and compared to the desired values. The property assessment is done on computer models of the scaffolds, and these models are based on micro-CT images. As a proof of principle, three porous scaffolds were evaluated with this protocol: stainless steel, hydroxyapatite, and titanium. These examples demonstrate that the modelling technique is able to quantify important scaffold properties. Thus, a powerful technique for automated screening of bone tissue engineering scaffolds has been developed that in a later stage may be used to tailor the scaffold properties to specific requirements.

Biocompatible Materials↗

PubMed2005

The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits.

BACKGROUND: Tissue-engineering techniques combined with gene therapy have been recently reported to improve osteogenesis. In this study, tissue-engineered bone constructed by human Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) was explored in an ectopic bone formation model in rabbits. METHODS: A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected in vitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the target gene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted to explore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined with Natural Non-organic Bone (NNB), a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x 3 mm, at a concentration of 5 x 10(7) cells/ml. They were subsequently implanted into 6 rabbits subcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation. RESULTS: pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, gene transfer efficiency reached more than 30%. Target gene transfer could strengthen the transcription of BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In the ectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in the bMSCs group was (17.2 +/- 7.1)%, and pEGFP group was (14.7 +/- 6.1)%, while pEGFP-hBMP-4 group was (29.5 +/- 8.2)%, which was the highest among the groups (F = 7.295, P < 0.01). CONCLUSIONS: The mammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transfer efficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoted differentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection of bMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bone tissue engineering applications.

Animals↗

PubMed2002

A novel pulsatile, laminar flow bioreactor for the development of tissue-engineered vascular structures.

Exposure of vascular cell-seeded, tubular, biodegradable polymers to pulsatile flow conditions has been proposed as a method to develop tissue-engineered blood vessels by "maturing" structural integrity, and increasing collagen content, suture retention, burst pressure, and tissue formation. These in vitro tissue-engineered arteries demonstrate contractile responses to pharmacologic agents and express markers of vascular differentiation. Current methods to induce pulsatile flow in a bioreactor system are limited by the creation of nonphysiologic pressure waveforms and noncompliant reservoirs to house the tissue-engineered vascular constructs. We have developed a novel method for the in vitro development of tubular vascular structures by using a mechanical ventilator to induce pulsatile, laminar flow into a fluid column, resulting in pressurized waveforms similar to mammalian physiology. The vascular constructs are housed in semicompliant tubing to facilitate an additional variable of circumferential stretch as a potential signaling mechanism. This approach more closely approximates mammalian physiology and we hypothesize that it will facilitate mechanical signaling necessary for the development of tissue-engineered vessels for clinical applications.

Bioreactors↗

PubMed2002

A novel small animal model of left ventricular tissue engineering.

BACKGROUND: Complex congenital cardiac anomalies involving ventricular hypoplasia require either staged palliative reconstruction, converting the circulatory system to a single ventricle based pump, or allogeneic transplantation. Tissue engineering offers the potential for complete reconstruction of these defects, but is limited by the inability to model myocardial tissue engineering in a small animal. Our goal was to develop a small animal model for ventricular tissue engineering using rat heterotopic heart transplantation. METHODS: Donor hearts were explanted after cardioplegic arrest and the left ventricular volume was augmented by the implantation of a biodegradable engineered construct. The heart was then transplanted heterotopically into syngeneic recipients creating either a volume loaded, functioning left ventricle, or a non-functioning left ventricle. Some of the engineered constructs were seeded with multipotent bone marrow-derived mesenchymal progenitor cells before implantation. Animals were evaluated by echocardiography, morphology, histology, and immunohistochemistry after 1 month. RESULTS: A scaffolding constructed from polytetrafluoroethylene, polylactide mesh, and type I and IV collagen hydrogel resulted in minimal intracardiac inflammation without aneurysmal dilatation. Successful transplantation and differentiation of mesenchymal progenitor cells was accomplished using this scaffolding. No ventricular arrhythmias resulted from this surgical manipulation and echocardiography revealed both end systolic and diastolic volume augmentation with ventricular expansion. CONCLUSION: We have developed an in vivo model of ventricular tissue engineering using heterotopic heart transplantation. Future work will focus on construction of ventricular tissue around pre-fabricated vascular networks in order increase cellular engraftment for ventricular reconstruction.

Animals↗