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Rezension / Literaturbericht, 2016
125 Seiten, Note: 1
EVENTS AND PROCESSES REQUIRED FOR PERIODONTAL REGENERATION
STRATEGIES TO ENGINEER TISSUE
TISSUE ENGINEERING TRIAD
MATRICES FOR TISSUE REGENERATION
CELL TYPES AND MOLECULES PARTICIPATING IN PERIODONTAL REGENERATION
REQUIREMENTS FOR SUCCESSFUL PERIODONTAL TISSUE ENGINEERING
GUIDED TISSUE REGENERATION:
RECEPTORS FOR GROWTH FACTORS
RATIONALE FOR THE USE OF GROWTH FACTORS IN PERIODONTAL REGENERATION
Periodontitis is the most common inflammatory disease in humans and forms the main cause of damage to periodontal tissues, which can finally lead to premature loss of teeth or even complete dentition, when left untreated. Conventional clinical treatment of periodontits involves the reduction or elimination of the periodontal pathogenic condition to stop the progression of the disease. Although this eradicates the clinical symptoms of the disease, it does not result in regeneration of the lost or destroyed tissue structures. Therefore current periodontal research is focused on the development of an approach that results in complete and predictable regeneration of the periodontal tissue. Achieving regeneration is challenging, as three periodontal tissues are involved, that is, the root cementum, periodontal ligament (PDL) and alveolar bone.1
The goal of tissue engineering and regenerative medicine is to promote healing and ideally true regeneration of a tissue’s structure and function more predictably, more quickly and less invasively than allowed by previous techniques.2 The term tissue engineering was originally coined to denote the construction in the laboratory of a device containing viable cells and biological mediators in a synthetic or biologic matrix that could be implanted in patients to facilitate regeneration of a particular tissue.3
Periodontal regeneration attributes to a complete recovery of the periodontal tissues in both height and function, that is the formation of alveolar bone, a new connective tissue attachment through collagen fibers functionally oriented on the newly formed cementum.4
The term Tissue Engineering was initially defined by the first National Science Foundation (NSF) sponsored meeting in 1988 as the “application of the principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationship in normal and pathological mammalian tissues and the development of biological substitutes for the repair or regeneration of tissue or organ function”. It differs from standard therapies in that engineered tissues become integrated within the patient, affording a potentially permanent and specific treatment of the disease state.5
In 1993, Langer and colleagues proposed tissue engineering as a possible technique for regeneration of lost tissue, and the restoration of various human tissue and organs is starting to become a reality.
The concept of tissue engineering was introduced originally to address the chronic shortage of donated organs. This approach reconstructs natural target tissue by combining three elements: a scaffolds or matrix, signaling molecules (for example, growth and differentiation factors and genes), and cells. Current approaches to tissue engineering can be divided roughly into two main types: ex vivo and in vivo. In the former, the target tissue is created in laboratory by culturing cells on biodegradable scaffolds in the presence of specific trophic factors before their transplantation into the body. In the latter approach the three elements mentioned above are placed into a tissue defect in situ and the tissue is restored by maximizing the natural healing capacity of the body by creating a local environment that is favorable for regeneration.6
Tissue engineering applies the principles of Biology, Chemistry, Physics and Engineering for the development of substitutes that replace, repair or enhance biological function of diseased and damaged human body parts, by manipulating cells via their extracellular microenvironment. This three dimensional extracellular architecture ("scaffold") can be fabricated in the shape of the tissue we want to restore, with the help of either polymer hydrogel, self-assembly, non- woven matrix, nano-fibrous electrospun matrices, 3D weaving, or any other textile technology-based techniques, depending upon their structural and functional requirements. This concept in periodontics began with guided tissue regeneration, a mechanical approach utilizing non-resorbable membranes to regenerate periodontal defects. In dental implantology, guided bone regeneration membranes, with or without mechanical support, are used for bone augmentation.5
Providing a history of tissue engineering it is inevitable to return to the basics, starting in 1665, when Hooke7(1635-1703) discovered small holes in cross-sections, which he called cells and described in his book Micrographia. In 1805, Oken8 stated, "All life is based on individual cells."
In 1838-1839, Schleiden9 and Schwann10 formulated the so-called "Cell Theory" based on their microscopic findings. This theory summarized their findings as:
- The cell is the unit of structure, physiology, and organization in living things
- The cell retains a dual existence as a distinct entity and a building block in the construction of organisms.
- Cells form by free-cell formation, similar to the formation of crystals (spontaneous generation).
In 1858, Virchow11(1821-1902) described his ideas about cell formation saying that cells arise from pre-existing cells, confuting Schleiden9and Schwann's10 idea of spontaneous cell generation. Virchow11 presented his ideas about regeneration stating that tissue regeneration is dependent on cell proliferation. This led research to focus attention toward the more fundamental cellular level. Thiersch12 attempted to grow skin cells into granulating wounds. In doing this, he discovered the important influence of granulation tissue on wound healing in 1874.
Loeb13 first reported the idea of growing cells outside the human body in 1897. From that point on many researchers experimented with growing cells in vitro. While experimenting with different media, survival was established, despite no growth. Harrison14 (1870-1959) was the first to grow frog ectodermal cells in vitro in 1907, thus developing the first neuronal tissue culture line. This was followed in 1912 by Carrel15 who was able to grow pieces of chick embryo in various media, which he initially maintained for 85 days, and subsequently for years. Later, interest arose in growing cells instead of complete tissues. In 1916, Rous and Jones16 discovered that trypsin is capable of degrading matrix proteins, thus separating cells. Trends changed toward expansion of cell types throughout the 1940s and soon after, much research was performed, which led to the ability to grow tissue-specific cell lines in vitro. Enders17 contributed greatly to the use of human embryonic cells in 1952.
In early 1970’s W.T.Green ,MD, undertook number of experiments to generate new cartilage using chondrocytes seeded onto spicules of bone and implanted in nude mice. Although unsuccessful, he correctly concluded that with the advent of innovative biocompatible material it would be possible to generate new tissue by seeding viable cells onto approximately configured scaffolds. Dr.Bruke and Yanas collaborated in studies in both the laboratory and in humans to generate a tissue engineered skin substitute using a collagen matrix to support the growth of dermal fibroblast. Dr. Howard Green later transferred sheets of keratinocytes onto burn patients, while D.Eugene Bell seeded collagen gels with fibroblasts, referring to them as concentrated collagen gels.
The term Tissue Engineering was initially defined by the first National Science Foundation (NSF) sponsored meeting in 1988 as the “application of the principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationship in normal and pathological mammalian tissues and the development of biological substitutes for the repair or regeneration of tissue or organ function”.18
In 1998, the development of stem cell lines formed the basis of modern tissue engineering.19 Research since 1998 has focused on identifying the differential capacities of embryonic and adult stem cells and how to influence and accompany this differentiating pathway.
Aside from this, research focuses on the problems associated with expansion of autologous and allogeneic differentiated cells, as a large number of cells are needed to generate a relatively small volume of tissue. Furthermore, fundamental research is continually being performed to gain insight into the cellular interactions that are of great importance in tissue engineering.20
REVIEW OF LITERATURE
For successful periodontal regeneration, protection of the healing site from the oral environment is required. Initial sealing and isolation of the site to prevent tissue damage induced by low-grade infections may dramatically improve the regenerative process. Whether this could be achieved by encouraging early epithelial attachment to the root surface (yet preventing its downgrowth) remains to be established. It would seem advantageous during the very early stages of periodontal wound healing to form a physiological epithelial seal and create a more favorable closed environment for predetermined stages of regeneration to occur.
Also, in order for periodontal regeneration to occur, many events have to be regulated at the cellular and molecular level.
The various events are21:
- Regeneration depends upon availability of appropriate cell types and signals that activate these cells.
- "Wrong'" cell types may have to be excluded
- Local environment plays a major role in the recruitment of the right cells and preventing the wrong cells. The local environment includes cementum matrix and cementodentinal junction.
- Substances in the local environment affect cell adhesion, proliferation and differentiation.
- The effect may be cell-specific and/or nonspecific.
Previous regenerative technologies as well as those in current use today show less than optimal outcomes for a number of reasons, including:
- inability to control the formation of a long junctional epithelium
-inability to adequately seal the healing site from the oral environment and prevent infection
inability to maintain the wound as a closed rather than open system .
-restriction of regeneration to the bone compartment and regenerative processes in the cementogenic and fibrous compartments;
-inability to define precisely the growth and differentiation factors needed for regeneration
-the possibility that growth factors may not be sufficiently discriminative in their ability to induce regeneration, and thus the induction of particular transcription factors as an earlier event of cell stimulation may be warranted
- infection of the implanted membrane or regenerative material postoperatively.
Successful tissue reconstruction requires the regeneration of cells and extracellular matrix in the correct spatial and functional relationship.
Tissue engineering can be accomplished in one of two ways:
a) By reconstruction of the tissue ex vivo followed by implantation into the desired site and its subsequent incorporation by surrounding host tissue,
b) By regeneration of the tissue in situ in the host.
In some cases, a single tissue, such as bone or cartilage, is the desired end point. In others several tissue types in functional apposition are required. An example of this latter end point is the periodontal environment, where bone, periodontal ligament, and the tooth root surface must coexist and operate together for optimal results.
Tissues are composed of cells, insoluble extracellular matrix (ECM), and soluble molecules that serve as regulators of cell function (generally polypeptides and eicosanoids). Using these three components of tissue, strategies to engineer tissue either in vitro or in vivo may be developed.2
There are advantages and disadvantages to engineering tissues in vitro versus in vivo. One advantage of in vitro strategies may be ability to examine the material as it is formed and to perform specific measurements prior to implantation. A disadvantage, however, particularly in the production of musculoskeletal tissue that must play a load-bearing role, is the absence of a physiologic mechanical environment during the formation of tissue in vitro. It is now well known that mechanical force serves as a critical regulator of cell function and can profoundly influence tissue architecture as it is forming. Because there is still much to be learned with regard to the mechanical environment that exists during bone formation in vivo, it is not yet possible to re-create such an environment in vitro during engineering of most tissues.2
Another disadvantage of the formation of musculoskeletal tissue outside of the body is the necessary incorporation of the tissue; after being implanted. This incorporation requires that the engineered tissue be mechanically coupled to the surrounding structures. Union of the implanted tissue with the host organ requires remodeling-degradation and new tissue formation-at the Page
interfaces of the implant with the host tissues. The fact that remodeling of the implanted tissue, engineered in vitro, is essential for its functional incorporation indicates the benefit to be derived from de novo regeneration in vivo, during which the incorporation occurs as the tissue is being formed.
Thus, for certain tissues (eg, musculoskeletal), an effective strategy may be to facilitate tissue formation in vivo, under the influence of the physiologic mechanical environment. However, one, disadvantage of this approach is that the regenerating tissue may be dislodged or degraded by the mechanical forces normally acting at the site, before the regenerating tissue is fully formed and incorporated .2
Langer and Vacanti (1993) 22 have explained three general strategies adopted for the creation of new tissue:
i) Isolated cells or cell substitutes: this approach avoids the complications of surgery, allows replacement of only those cells that supply the needed function, and permits manipulation of cells before infusion.
ii) Tissue inducing substances: the success of this approach depends on the purification and
large scale production of appropriate signal molecules, such as growth factors and in many cases the development of methods to deliver these molecules to their targets.
iii) Cell placed on or within matrices: in closed systems, the cells are isolated from the body by a membrane that allows permeation of nutrients and wastes but prevent large entities such as antibodies or immune cells from destroying the transplant. These systems can be implanted or used as extra corporeal devices. In open systems, cells attached to matrices are implanted and become incorporated into the body.
These strategies can be categorized into three major classes: conductive, inductive, and cell transplantation approaches. These approaches all typically utilize a material component, although with different goals.
Conductive approaches utilize biomaterials in a passive manner to facilitate the growth or regenerative capacity of existing tissue. An example of this is very familiar to dentists, and particularly periodontists, is the use of barrier membranes in guided tissue regeneration. 23
Nyman et al (1982) 24,25 were the first to successfully use osteoconductive mechanisms in providing a means for selective wound healing by supporting the ingrowth of the periodontal supporting cells, while excluding gingival epithelial and connective tissue cells from reconstruction sites. Techniques and materials are still being optimized in guided tissue regeneration. However, the appropriate use of barrier membranes promotes predictable bone repair and histologically verifiable new attachment with new formation of cementum and periodontal ligament fibers.
The second major tissue engin e ering strategy (induction) involves activating cells in close prox-imity to the defect site with specific biological signals. The origins of this mechanism are rooted in the discovery of bone morphogenetic proteins (BMPs). 23
Urist (1965) 26 was the first to show that new bone could be formed at nonmineralizing, or ectopic, sites after implantation of powdered bone (bone demineralized and ground into fine particles). Contained within the powdered bone were proteins (BMPs), which turned out to be the key elements for inducing bone formation.
These proteins are now available in recombinant forms and produced on a large scale by biotechnology companies. BMPs have been used in many clinical trials and are very promising as a means of therapy and supplementation in the regeneration and repair of bone in a variety of situations, including nonhealing fractures and periodontal disease.
Recent tissue engineering approaches have been concerned with more accurately mimicking the processes of embryonic development and wound healing. Inductive approaches to engineering tissues are aimed at manipulating the process of tissue formation by controlled delivery of various bioactive factors involved in developmental processes.
These factors can provide the means for manipulating cell proliferation, chemotaxis, differentiation and matrix synthesis, and thus exhibit potential for regenerative medicine. Factors examined as possible directors of tissue development include polypeptide growth factors and deoxyribonucleic acids (DNA) that encode for bioactive factors.27
Regeneration of lost tissues shall require the recruitment of cells that have the potential to differentiate into specialized regenerative cells.
It also requires a scaffold or a supportive template which is necessary for the organization of these replicating cells. And in addition, it requires the presence of certain signaling molecules which act as growth and differentiating factors.
Thus, the tissue engineering triad combines three key elements, i.e., the scaffolds (matrices), signaling molecules (growth factors), and cells. By combining these three elements in appropriate environment tissue regeneration can often be accomplished, time being an important factor.2
Abbildung in dieser Leseprobe nicht enthalten
Matrices for engineering bone and soft connective tissues have included synthetic and natural calcium phosphates and myriad synthetic (e.g., polylactic acid and polyglycolic acid) and natural polymers (e.g., collagen and fibrin). Material to be used for the fabrication of matrices to engineer tissue in vitro, or to facilitate regeneration in vivo, must have the microstructure and chemical composition required for normal cell growth and function. For bone regeneration, a material possessing similar physical, chemical, and mechanical properties is desirable since all of these properties will influence normal bone cell growth and function.
Roles in the Regenerative Process
A matrix can play several roles during the process of regeneration in vivo:2
1. It can structurally reinforce the defect site so as to maintain the shape of the defect and prevent distortion of surrounding tissue. For example, cysts that form in the subchondral bone underlying the articulating surfaces of joints can lead to collapse of the joint surface.
2. The matrix can serve as a barrier to the ingrowth of surrounding tissue that may impede the process of regeneration. The concept of guided tissue regeneration is based in part on the prevention of overlying gingival tissue from collapsing into the periodontal defect.
3. The matrix can serve as a scaffold for migration and proliferation of cells in vivo or for cells seeded in vitro.
4. The matrix can serve as an insoluble regulator of cell function through its interaction with certain integrins and other cell receptors.
Tissue engineering scaffolds are highly engineered structures that accommodate cells, facilitate their expression and resorb to facilitate regeneration of tissue.
Porous polymer scaffolds are promising materials for tissue engineering because they offer a physical, three-dimensional (3D) support and serve as a template for cell proliferation and ultimately tissue formation. The scaffolds may vary in their compositions (coral, collagen. and synthetic polymers), but the desirability of having cells attach to the scaffold material is a common goal.28
Pore sizes of greater than 300 mm were observed to have a greater penetration of mineralised tissue in comparison with smaller pore sizes. At pore sizes of 75 mm. hardly any mineralised tissue was found within the scaffold. It is believed that for smaller pore sizes the penetration of neovascularisation, and hence nutrient supply, to the growing cells is hindered.28 Pores needed to be greater than 100 mm for bone tissue ingrowth. Studies on corals revealed that pore connectivity was necessary for deeper tissue growth within the porous body. These channels allowed the supply of oxygen and nutrients to cells inside the scaffold and facilitated waste transfer out of the scaffold. The requirement of pore size and connectivity were thus quickly established for scaffolds.
There are several requirements in the design of scaffolds for tissue engineering. Many of these requirements are complex and not yet fully understood.
Scaffolds designed for use in cell-based therapies to repair/regenerate bone should provide the following minimal requirements.28
a. A three-dimensional (3D) and highly porous structure to support cell attachment, proliferation and extra-cellular matrix (ECM) production
b. An interconnected/permeable pore network to promote nutrient and waste exchange
c. A biocompatible and bioresorbable substrate with controllable degradation rates
d. A suitable surface chemistry for cell attachment, proliferation, and differentiation
e. Mechanical properties to support, or match, those of the tissues at the site of implantation
f. An architecture which promotes formation of the native anisotropic tissue
g. A reproducible architecture of clinically relevant size and shape.
To date, most scaffolds reported for bone repair conform to only a few of these criteria.
Zhang and Ma (1997) 29 describes the preparation and morphologies of three-dimensional porous composites from poly (L-lactic acid) (PLLA) or poly (D,L-lactic acid-co-glycolic acid) (PLGA) solution and hydroxyapatite (HAP). A thermally induced phase separation technique was used to create the highly porous composite scaffolds for bone-tissue engineering. Freeze drying of the phase-separated polymer/HAP/solvent mixtures produced hard and tough foams with a co-continuous structure of interconnected pores and a polymer/HAP composite skeleton. The microstructure of the pores and the walls was controlled by varying the polymer concentration, HAP content, quenching temperature, polymer, and solvent utilized. The porosity increased with decreasing polymer concentration and HAP content. Pore sizes ranging from several microns to a few hundred microns were obtained. The composite foams showed a significant improvement in mechanical properties over pure polymer foams. They are promising scaffolds for bone-tissue engineering.
Shoichet et al (1999) 30 established the percentage of cells anchoring to polymer scaffolds as a function of initial cell seeding density and then investigated bone tissue formation throughout scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 × 106 cells/cm3 were seeded onto 3D scaffolds. After 1 hour in culture, it was determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 × 106 cells/cm3 were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 × 106 cells/cm3 showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 × 106 cells/cm3 were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization,
occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.
Ganta et al (2003) 31 performed a study to develop nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 micron in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous micro texture can be achieved, this matrix may be useful in many tissue-engineering applications.
Zigang Ge et al (2003) 32 demonstrates the potential of HA-chitin matrixes as a good substrate candidate for tissue engineered bone substitute. Hydroxyapatite (HA) in 25%, 50% and 75% w/w fractions was incorporated into chitin solutions and processed into air- and freeze-dried materials. These HA-chitin materials were exposed to cell cultures and implanted into the intra musculature of a rat model. The HA-chitin materials were found to be non-cytotoxic and degraded in vivo. The presence of the HA filler enhanced calcification as well as accelerated degradation of the chitin matrix. The freeze-dried HA-chitin matrixes were selected for further cell seeding experiments because of their porous nature. Mesenchymal stem cells (MSCs) harvested from New Zealand White rabbits were induced into osteoblasts in vitro using
dexamethasone. These osteoblasts were cultured for 1 week, statically loaded onto the porous HA-chitin matrixes and implanted into bone defects of the rabbit femur for 2 months. Histology of explants showed bone regeneration with biodegradation of the HA-chitin matrix. Similarly, green fluorescence protein (GFP) transfected MSC-induced osteoblasts were also loaded onto porous HA-chitin matrixes and implanted into the rabbit femur. The results from GFP-transfected MSCs showed that loaded MSCs-induced osteoblasts did not only proliferate but also recruited surrounding tissue to grow in.
Lee et al (2003) 33 Hybrid scaffolds composed of b-chitin and collagen were prepared by combining salt-leaching and freeze-drying methods. The chitin scaffold used as a framework was easily formed into desired shapes with a uniformly distributed and interconnected pore structure with average pore size of 260-330 mm. The mechanical strength and the rate of biodegradation increased with the porosity, which could be modulated by the salt concentration. In addition, atelocollagen solution was introduced into the macropores of the chitin scaffold to improve cell attachment. The fibroblasts showed a good affinity to and proliferation on all collagen-coated chitins.
Many methods to prepare porous three-dimensional biodegradable scaffolds have been developed in tissue engineering, including:
- gas forming,
- fiber extrusion and bonding,
- three-dimensional printing,
- phase separation,
- emulsion freeze-drying
- porogen leaching.
Recently, a new kind of hybridization technique was developed and used to fabricate porous hybrid scaffolds by combining synthetic and natural biodegradable polymers such as poly (D.L-lactic-co-glycolic acid) (PLGA) and collagen. In the hybrid scaffold, the synthetic biodegradable polymers are easily formed into desired shapes with good mechanical strength and the duration of degradation can be estimated. Despite these advantages, the scaffolds derived from synthetic
polymers are insufficient for cell-recognition signals and their hydrophobic properties obstruct smooth cell seeding. In contrast, naturally derived polymers have the potential advantages of specific cell interactions and a hydrophilic nature, but possess poor mechanical properties. Thus, these two kinds of biodegradable polymers have been hybridized to combine the advantageous properties of both constituents. Furthermore, hybridization with collagen facilitates cell seeding and spatial cell distribution, and promotes cell immigration and neo-angiogenesis.
Kruyt et al (2003) 34 investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, they investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, they evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40μm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 m, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 m. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.
Yang C et al (2004) 35 Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions.
The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.
Di Martino et al (2011) 36 Tissue engineering aims to regenerate native tissues and will represent the alternative choice of standard surgery for different kind of tissue damages. The fundamental basis of tissue engineering is the appropriate selection of scaffolds and their morphological,
mechanical, chemical, and biomimetic properties, closely related to cell lines that will be seeded therein. The aim of this review is to summarize and report the innovative scientific contributions published in the field of orthopedic tissue engineering, in particular about bone tissue engineering. We have focused our attention on the electrospinning technique, as a scaffold fabrication method. Electrospun materials are being evaluated as scaffolds for bone tissue engineering, and the results of all these studies clearly indicate that they represent suitable potential substrates for cell-based technologies
Lee et al (2014) 37 In this study, they utilized an electrospinning (ELSP) technique to design a novel wound dressing. Chitosan (CTS) nanofibers containing various ratios of silver nanoparticles (AgNPs) were obtained. AgNPs were generated directly in the CTS solution by using a chemical reduction method. The formation and presence of AgNPs in the CTS/AgNPs composite was confirmed by x-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV) and thermogravimetric analysis (TGA). The electrospun CTS/AgNPs nanofibers were characterized morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These nanofibers were subsequently tested to evaluate their antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa) and gram-positive Methicillin-resistant Staphylococcus aureus (MRSA). Results of this antibacterial testing suggest that CTS/AgNPs nanofibers may be effective in topical antibacterial treatment in wound care.
The cell types required for periodontal regeneration include epithelial cells to seal the wound area, fibroblastic cells for the soft connective tissues of the gingiva and periodontal ligament, mineralized tissue-forming cells (alveolar bone cells) for bone formation and endothelial cells for forming blood vessels.21
During the regenerative process, these cells must interact with a variety of soluble mediators such that the course of regeneration is dictated by a combination of molecule-cell, cell-matrix and cell-cell interactions. Very little is known about the signals that initiate and regulate these interactions in vivo.
Soluble mediators of importance include growth factors, cytokines, attachment or adhesion proteins and structural components.
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Exogenous Cells for engineering Tissues
In many tissues such as bone, the number and mitotic activity of precursor cells is so high that there is normally an ample source of endogenous cells to populate implanted scaffolds for the regeneration of tissue. In most circumstances, matrices alone can serve to facilitate regeneration. Exogenous cells and mitogenic factors may only be necessary in special cases in these tissues, when the proliferation of the precursor cells is impeded or their pool has been greatly diminished by previous surgery or concomitant disease. When there are indications for exogenous cells or soluble regulators, their incorporation into implantable matrices is generally required to ensure their localization at the treatment site. An alternative is to inject a cell suspension into a sealed compartment containing the defect.
Currently, allogeneic and autologous cells from the tissue to be regenerated, grown in culture from harvested tissue that has been digested in vitro are being investigated for the regeneration of a variety of tissues. There are advantages and disadvantages to each approach. When allogeneic cells are used, the potential for immune response and disease transmission must be considered. Issues related to the use of autologous cells include the requirements for harvesting the cells and donor site morbidity. Recently, it has been proposed that marrow stromal stem cells can be used as precursors for many connective tissues. However, it has not yet been demonstrated that the conditions for differentiation of these cells can be reliably implemented and the desired differentiation state can be maintained.2
Van Dijk et al (1991) 38 conducted a study to test the hypothesis that seeded periodontal ligament cells are able to create new attachment. In one beagle dog, a premolar was removed and scrapings of the ligament were cultured. Artificial periodontal defects were made and cultured ligament cells were seeded on the planed root surfaces and covered with mucoperiosteal flaps. The opposite side served as control. After 4 months, the dog was sacrificed and histological and electron microscopical sections were prepared. The seeded root surfaces were almost completely covered with cementoblasts, whereas in controls, epithelial down growth could be observed. They concluded that seeding of ligament cells could be a promising technique to create new connective tissue attachment.
Feng F & Hou LT (1992) 39 conducted a study to examine the healing of periodontal defects using HA grafts coated with fibroblasts isolated from the periodontal ligament (PDL). They cultured fibroblast-like cells either from a clinically healthy site of the PDL or gingival tissues of the subject receiving the cell transplantation. They added HA to the cultures and allowed the cells to migrate onto the HA surface. Then the HA particles coated with cells were harvested and transplanted into the periodontal osseous defects of four patients after phase I treatment. Three HA grafts without coating cells were used as controls. Periapical x-ray and clinical parameters were monitored for up to six months. Scanning electron microscopy demonstrated that fibroblast-like cells had proliferated on the HA particles in vitro. Results showed that the
experimental group had greater pocket reduction and clinical attachment gain, and less gingival recession than did the control group at six months postoperatively. Periapical films revealed good filling of osseous defects in both groups. This study introduces a new biological approach for bringing PDL cells into intimate contact with root surfaces, in order to facilitate earlier repopulation of root surfaces with regenerative PDL cells.
Wakitani S et al (1994) 40 Osteochondral progenitor cells were used to repair large, full-thickness defects of the articular cartilage that had been created in the knees of rabbits. Adherent cells from bone marrow, or cells from the periosteum that had been liberated from connective tissue by collagenase digestion, were grown in culture, dispersed in a type-I collagen gel, and transplanted into a large (three-by-six-millimeter), full-thickness (three-millimeter) defect in the weight-bearing surface of the medial femoral condyle. The contralateral knee served as a control: either the defect in that knee was left empty or a cell-free collagen gel was implanted.
The periosteal and the bone-marrow-derived cells showed similar patterns of differentiation into articular cartilage and subchondral bone. Specimens of reparative tissue were analyzed with use of a semiquantitative histological grading system and by mechanical testing with employment of a porous indenter to measure the compliance of the tissue at intervals until twenty-four weeks after the operation. There was no apparent difference between the results obtained with the cells from the bone marrow and those from the periosteum. As early as two weeks after transplantation, the autologous osteochondral progenitor cells had uniformly differentiated into chondrocytes throughout the defects.
This repair cartilage was subsequently replaced with bone in a proximal-to-distal direction, until, at twenty-four weeks after transplantation, the subchondral bone was completely repaired, without loss of overlying articular cartilage. The mechanical testing data were a useful index of the quality of the long-term repair. Twenty-four weeks after transplantation, the reparative tissue of both the bone marrow and the periosteal cells was stiffer and less compliant than the tissue derived from the empty defects but less stiff and more compliant than normal cartilage.
Clinical Relevance: The current modalities for the repair of defects of the articular cartilage have many disadvantages. The transplantation of progenitor cells that will form cartilage and bone offers a possible alternative to these methods. As demonstrated in this report, autologous, bonemarrow-derived, osteochondral progenitor cells can be isolated and grown in vitro without the loss of their capacity to differentiate into cartilage or bone. Sufficient autologous cells can be generated to initiate the repair of articular cartilage and the reformation of subchondral bone. The repair tissues appear to undergo the same developmental transitions that originally led to the formation of articular tissue in the embryo.
Wang et al (2000) 41 Most of normal human somatic cells can divide only a finite number of times and inevitably become senescent. Telomerase is an enzyme that imparts replicative immortality by maintaining the length of the telomeres when expressed in reproductive and cancer cells. Cells that are mortal do not express the telomerase. Recently it was reported that the life span of the normal human cells could be successfully extended by introduction of telomerase into these cells. According to them fibroblasts exhibited an osteogentic potential, and therefore, can be considered as a type of "seed cells" in tissue engineering for bone repairing and reconstruction. But this potential was impaired by the limitation in life span and proliferative capacity of the normal fibroblasts.
Plasmid pGRN145 bearing a cDNA insert of human telomerase reverse transcriptase (hTERT) was introduced into the fibroblasts with osteogenic potential by electroporation. The stable hTERT+ fibroblast clones was established and cultured for long-term in a medium containing hygromycin-B. The exogenous hTERT mRNA expression and telomerase activity were detected. The hTERT+ fibroblasts showed shorter population doubling time and no beta-galactosidase stain, which indicated a stronger proliferative capacity and fewer signs of cell senescence, compared to their hTERT- counterpart.
This showed that the life span of hTERT+ fibroblasts was extended. The assays for DNA euploidy by flow cytometry and chromosome karyotype by cytogenetic technique showed no signs of heteroploidy, providing the data for cell carcinogenesis and utilization safety. The results of the present study suggested that the introduction of hTERT could make the life span of normal fibroblast extended without causing their malignant transformation, and such type of "longevous" fibroblasts might be clinically useful in tissue engineering for bone repairing and reconstruction.
Ou L, Liu H, Pang J (2000) 42 did a study in dogs to evaluate the effect of autogenous periodontal ligament cell transplantation in periodontal tissue regeneration. Periodontal ligament cells derived from the same dog were cultured with alpha-MEM. 1 x 10(5) cells of first passage were allowed to attach to the collagen membrane for 24 hours. The membrane-cells were transplanted into periodontal defect in the same dog. Then the defects were covered with e-PTFE membranes. The defects covered only with e-PTFE were the controls. Eighteen teeth of 6 dogs for each group were included. The dogs were sacrificed after 6 weeks. The results showed that new bone formation (4.00 +/- 0.13) mm in test group was significantly higher than that of in control group (3.09 +/- 0.28) mm, P < 0.05. The new cementum formation in test group was better than control group (P < 0.05). The results suggested that periodontal ligament cells transplantation with guided tissue regeneration technique could enhance periodontal tissue regeneration in dogs.
Yang ZM, Huang FG, Qin TW (2002) 43 in a clinical trial treated various types of bone defect using Bio-derived tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. All the cases were followed up after operation, averaged in 18.5 months. No obvious abnormalities were observed. They concluded that Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.
Dogan et al (2003) 44 performed a study to assess the seeding of fibroblast-like cells to promote periodontal healing in artificial fenestration defects in a dog. Fibroblast-like cells were cultured by incubating regenerated periodontal ligament tissue, which had been surgically taken, underneath a Teflon membrane. Fenestration defects were surgically induced on the maxillary canine and first molar teeth at a spacing of 5 to 5 mm. Passage 4 cells (2 x 10(5) cells) in autologous blood coagulum were placed on root surfaces in two defects; the remaining two defects were used as controls. Healing was evaluated histomorphometrically on postoperative day 42. The main periodontal healing pattern consisted of connective tissue adaptation in three of the four specimens including one control, with cementum formation at 9-12%; one control specimen that exhibited 100% cementum formation. New bone formation was greater in the cell-seeding group (84%) compared with control (39%). In the cell-seeding group, one specimen exhibited total regeneration of bone (100%); however, the connective tissue located between newly formed bone and the root surface was observed to adapt to the dentin surface, with limited cementum formation. Seeding of cells from periodontal ligament may be promising to promote periodontal regeneration, but needs to be investigated in further studies.
Saito et al (2003) 45 performed a study to examine the periodontal healing after transplantation of teeth with reduced periodontal ligament that had been cultured in vitro. Twenty-five incisors from four beagles were used. After the teeth were extracted, the periodontal ligament and cementum were removed from coronal part of the roots and the roots were planed. The periodontal ligament of the apical part was retained. Fourteen teeth of the experimental group were transplanted following culture for 6 weeks. Eleven teeth of the control group were similarly prepared and immediately transplanted without tissue culture. Four weeks after transplantation, the specimens were prepared for histological analysis. Downgrowth of junctional epithelium on the root of experimental group was significantly less than control. Most of the root planed surfaces of experimental group were covered with periodontal ligament fibers oriented parallel or inclined to the root surfaces and limited new cementum formation was observed near the apical end of the planed root. There was no significant difference between groups in observations on the root surface with remaining periodontal ligament. From the above results, it was concluded that periodontal tissue culture of teeth with root planed surface and remaining periodontal ligament could reduce the extent of epithelium downgrowth and increase connective tissue adhesion on the root planed surface, as well as minimize damage to remaining periodontal ligament, after transplantation of teeth.
According to Xiao et al (2003) 46 in their study, collagen type I matrices seeded with cells with osteogenic potential were implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with
osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.
Mesenchymal stem cells:
Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, one expected mesenchymal stem cells to differentiate into periodontal ligament.
Mesenchymal stem cells or human bone marrow stromal stem cells are defined as pluripotent progenitor cells with the ability to generate cartilage, bone, muscle, tendon, ligament and fat. These primitive progenitors exist postnatally and exhibit stem cell characteristics, namely, low incidence and extensive renewal potential. These properties in combination with their developmental plasticity have generated tremendous interest in the potential use of mesenchymal stem cells to replace damaged tissues. In essence mesenchymal stem cells could be cultured to expand their numbers then transplanted to the injured site or after seeding in/on shaped biomimetic scaffold to generate appropriate tissue constructs.47
Stem cells are cells that, in cell cultures at least, have the ability to divide forever. They also have the capacity to develop into specialized populations of cells. There are stem cells in developing embryos, and in recent years, scientists have confirmed the existence of stem cells in adult humans. Recent data suggest that stem cells are not only active in embryos, but act throughout our lives, replacing worn and damaged mature cells. Before 1998, when the first stem cells were actually identified, they were merely assumed to exist. As evidence for their existence, researchers cited bone marrow transplants. In the treatment of certain cancers, chemotherapy destroyed all of the cells of the bone marrow. That treatment was followed by transplants of bone marrow from healthy donors. The small volume of transplanted bone marrow eventually gave rise to enough cells to repopulate the body with red blood cells, white blood cells and platelets. The source of all of those cells was presumed to be stem cells, and that presumption has proved correct.
Thus, an alternative approach for skeletal repair is the selection, expansion and modulation of osteoprogenitor cells in combination with a conductive or inductive scaffolds to support and guide regeneration together with judicious selection of osteotropic growth factors . 47
Stem cells are best understood in terms of how committed they are to becoming any particular type of cell. The categories into which they fall include:
- Totipotent stem cells
- Pluripotent stem cells
- Multipotent stem cells
- Adult stem cells
Totipotent stem cells
Human cells can be divided into sex or germ cells (eggs and sperm) and somatic cells (all of the rest of our cells). When a sperm cell and an egg cell unite, they form a one-celled fertilized egg. This cell is totipotent, which means that it has the potential to give rise to any and all human cells, such as brain, liver, blood or heart cells. The first few cell divisions in embryonic development produce more totipotent cells. After four days of embryonic cell division, the cells begin to specialize.
Pluripotent stem cells
On the fourth day of embryonic development, the ball of cells forms itself into an outer layer, which will become the placenta, and an inner mass, which will form the tissues of the developing human body. These inner cells, though they can form nearly any human tissue, cannot do so without the outer layer, and so are not totipotent, but pluripotent. As these pluripotent stem cells continue to divide, they begin to specialize further.
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