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Biomedical subjects

Michael Sittinger

Publications and source records attributed to Michael Sittinger.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression profiling of human articular cartilage grafts generated by tissue engineering.

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. In this study autologous human cartilage tissue engineering grafts based on bioresorbable polyglactin/polydioxanone scaffolds were analyzed on the broad molecular level. RNA from freshly isolated, primary and expanded adult articular chondrocytes and from three-dimensional cartilage grafts were used for gene expression profiling using oligonucleotide microarrays. The capacity of cartilage grafts to form cartilage matrix was evaluated after subcutaneous transplantation into nude mice. Gene expression profiling showed reproducibly the regulation of 905 genes and documented that chondrocytes undergo fundamental changes during cartilage tissue engineering regarding chondrocyte metabolism, growth, and differentiation. Three-dimensional assembly of expanded, dedifferentiated chondrocytes initiated the re-differentiation of cells that was accompanied by the reversal of the expression profile of multiple players of the transforming growth factor (TGF) signaling pathway including growth and differentiation factor-5 and inhibitor of differentiation-1 as well as by the induction of typical cartilage-related matrix genes such as type II collagen and cartilage oligomeric matrix protein. Cartilage grafts formed a cartilaginous matrix after transplantation into nude mice. Three-dimensional tissue culture of expanded articular chondrocytes initiates chondrocyte re-differentiation in vitro and leads to the maturation of cartilage grafts towards hyaline cartilage in vivo.

Base Sequence↗
PubMed2006

Treatment of articular cartilage defects in horses with polymer-based cartilage tissue engineering grafts.

The objective of our study was to evaluate the integration of autologous cartilage tissue engineering transplants based on resorbable polyglactin/polydioxanone scaffolds into full-thickness cartilage defects of horses. Cartilage biopsies were taken from the non-load-bearing area of the lateral talus of the left tibiotarsal joint of eight healthy Haflinger horses. Tissue engineering cartilage transplants were generated by three-dimensional arrangement of autologous chondrocytes in biocompatible and resorbable polymer scaffolds. Full-thickness cartilage defects of 8 mm in diameter were created in the tubular bone condyle of the fetlock joint and cartilage grafts were fixed using an anchor system, while defects without grafting served as controls. After 6 and 12 months the repair tissue was evaluated histologically and showed formation of a cartilaginous tissue and good integration into the surrounding host tissue with firm bonding of the graft to the adjacent cartilage and the underlying subchondral bone. Biochemical analysis demonstrated that the content of glycosaminoglycans and hydroxyproline is comparable in repair tissue derived from treated and control defects. The use of three-dimensional autologous cartilage transplants based on resorbable polymer scaffolds ensures secure fixation, good integration of the graft into cartilage lesions, and is therefore suggested as a promising therapeutic option for the treatment of cartilage defects.

Animals↗
PubMed2006

Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum.

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.

Biocompatible Materials↗
PubMed2005

The morphology and biomechanical characteristics of subcutaneously implanted tissue-engineered human septal cartilage.

The purpose of the study was to examine the morphology and biomechanical characteristics of in vivo cultured tissue-engineered human septal cartilage as a prospective autogenous transplant material for subcutaneous implantation in reconstructive procedures. Chondrocytes were enzymatically isolated from human septal cartilage biopsies. The cell number was expanded in monolayer culture. Chondrocytes were then fixed on a non-woven poly-lactide-poly-glycolide (PGLA) polymer scaffold by means of fibrin glue. The PGLA-polymer construct was implanted subcutaneously on the back of athymic mice and allowed to mature for 6 or 12 weeks. After killing the mice, the formed cartilage was tested on a material testing machine with a highly standardized reproducible setting. Biomechanical testing consisted of an indentation test, which revealed the failure load and compressive modulus of the neocartilage. The failure load shows the upper limit of supported stress. The compressive modulus is a measure of the templates' stiffness. After testing, the templates were histologically stained. Native human septal cartilage served as a control group. Histological and macroscopic examination showed cartilage formation of a hyaline-like morphology. Histological staining revealed the synthesis of abundant mucopolysaccharid matrix. The biomechanical characteristics of neocartilage proved to be of no statistical difference compared to native human septal cartilage. The failure load and compressive modulus were initially somewhat lower and reached the control group's results after 12 weeks in-vivo. Summarizing, tissue engineered nasal cartilage matches typical mechanical characteristics of native hyaline cartilage. Its elasticity and failure load are of sufficient quality to meet the clinical requirements for reconstructive surgery.

Animals↗
PubMed2005

Chitosan: a versatile biopolymer for orthopaedic tissue-engineering.

Current tissue engineering strategies are focused on the restoration of pathologically altered tissue architecture by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable attention has been given to chitosan (CS)-based materials and their applications in the field of orthopedic tissue engineering. Interesting characteristics that render chitosan suitable for this purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be molded in various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. Due to its favorable gelling properties chitosan can deliver morphogenic factors and pharmaceutical agents in a controlled fashion. Its cationic nature allows it to complex DNA molecules making it an ideal candidate for gene delivery strategies. The ability to manipulate and reconstitute tissue structure and function using this material has tremendous clinical implications and is likely to play a key role in cell and gene therapies in coming years. In this paper we will review the current applications and future directions of CS in articular cartilage, intervertebral disk and bone tissue engineering.

Animals↗
PubMed2005

Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model.

OBJECTIVE: The aim of this study was to evaluate the growth characteristics of freshly isolated porcine chondrocytes in high-density pellet cultures and to preliminary investigate their use in an interactive in vitro model with synovial fibroblast cell lines to study rheumatoid arthritis (RA). DESIGN: 1.8x10(6) chondrocytes/cm2 were seeded in 48-multiwell plates. Thickness, cell number and cell distribution in pellet cross sections were documented over a 22-day-long period. Alcian blue staining, type I and type II collagen staining, real-time reverse transcriptase polymerase polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC) were used to characterize cartilage extracellular matrix (ECM) formation, and cell proliferation was demonstrated by Ki67 staining. Furthermore, 2-week-old chondrocyte pellets were co-cultured for additional 2 weeks with two human synovial fibroblast cell lines derived from a normal donor (non-invasive cell line) and a RA patient (invasive-aggressive (IA) cell line), respectively. RESULTS: Chondrocyte pellets from 11 individual preparations showed a significant increase in pellet thickness from 44+/-19 microm (day 3) to 282+/-19 microm (day 22). Calculation of chondrocyte distribution, cell number and pellet thickness indicated that pellet growth was due to ECM formation and not cell proliferation. This was also confirmed by low numbers of Ki67 positive chondrocytes and absence of cell clusters. HPLC, messenger RNA-analysis, histochemistry and antibody staining verified the expression of ECM components such as type II collagen, whereas type I collagen expression was very low. In contrast to the non-aggressive synovial fibroblast cell line, the IA synovial fibroblast cell line clearly showed cartilage invasion. CONCLUSION: Pellet formation of freshly isolated chondrocytes followed a reproducible developmental kinetics and showed typical immature hyaline cartilage properties. Such uniform cartilage pellets are very useful as a substrate for interactive cell culture models that simulate diseases like RA.

Animals↗
PubMed2004

Changes in the gene expression pattern of cytokeratins in human respiratory epithelial cells during culture.

The replacement of extensive tracheal defects resulting from intensive care medicine, trauma or large resections is still challenged by the re-epithelialization of an autologous or alloplastic trachea replacement. Therefore, this study was performed to investigate the potential of culture-expanded human respiratory epithelial cells (hREC) to regenerate a functional epithelium for tracheal tissue engineering. hREC from seven male nasal turbinates were freshly isolated, expanded on a collagenous matrix and subsequently cultured in high-density multi-layers to allow epithelial differentiation. The composition of epithelial cells in native respiratory epithelial tissue and culture-expanded hREC was analyzed by histological staining with Alcian blue and by immunohistochemical staining of cytokeratin pairs CK1/10 and CK5/14 with the antibodies 34betaE12 and CD44v6. Differentiation of culture-expanded hREC was further characterized by gene expression analysis of cytokeratins CK5, CK13, CK14 and CK18 using semi-quantitative real-time RT-PCR technique. Histological and immunohistochemical staining of culture-expanded hREC demonstrated basal cells covering the collagenous matrix. These cells formed a cellular multi-layer, which was composed of a basal layer of undifferentiated basal cells and an upper layer of cells differentiating along the squamous metaplasia and ciliated cell lineage. Lineage development of culture-expanded hREC was further documented by the induction of cytokeratins CK13 and CK18. Our results suggest that culture-expanded hREC have the potential to colonize collagen-coated biomaterials and to regenerate epithelial cell types for tracheal tissue engineering.

Adult↗
PubMed2004

Creating artificial perichondrium by polymer complex membrane macroencapsulation: immune protection and stabilization of subcutaneously transplanted tissue-engineered cartilage.

Functional organ or tissue failure is one of the most frequent, devastating and costly problems in modern health care. The field of tissue engineering has tremendous potential for developing new functional tissue. In reconstructive surgery, cartilage engineering could be a serious alternative to the established method of autologous cartilage transplantation. Recent studies demonstrate cartilage engineering by subcutaneous implantation of chondrocyte-seeded PGA/PLA-fibrin glue scaffolds in the backs of nude mice. In both autologous cartilage transplantation and cartilage engineering, the host immune response affects transplant integrity and cartilage morphology to an unforeseeable extent. To investigate whether polyelectrolyte complex (PEC) membranes can prevent rejection of cartilage transplants without neglecting tissue metabolism, tissue-engineered cartilage encapsulated with a PEC membrane was subcutaneously implanted in the backs of nude mice. Non-encapsulated tissue-engineered cartilage was used for the control group. Histochemistry and scanning electron microscopy were performed 4 and 12 weeks after implantation. There was no interaction between the host and the implant with an intact PEC membrane. With protection by PEC encapsulation, implanted tissue-engineered cartilage showed no signs of degeneration and had a significantly weaker cellular immune response than without it. Thus, PEC membrane encapsulation appears to be a novel approach for protecting cartilage implants from host immune response after autologous transplantation.

Animals↗
PubMed2004

Does low-intensity pulsed ultrasound stimulate maturation of tissue-engineered cartilage?

Traumatic events are a primary cause of local lesions of articular cartilage. Tissue engineered, cartilage-like structures represent an alternative to current treatment methods. The time necessary for tissue maturation and the mechanical quality of the regenerate at implantation are both critical factors for clinical success. Low-intensity pulsed ultrasound has proven to accelerate chondrogenesis in vitro. The goal of this study was to evaluate whether low-intensity pulsed ultrasound is capable of accelerating the process of cartilage maturation and increasing regenerate stability. Hyaline-like cartilage specimens were generated in vitro and subcutaneously implanted in the backs of nude mice. Twenty-eight animals received 20 min of low-intensity pulsed ultrasound treatment daily, and 28 animals received a sham treatment. Specimens were explanted after 1, 3, 6, and 12 weeks, mechanically tested with the use of an indentation test, histologically examined, and processed for RT-PCR. The Young's moduli significantly increased from 3 to 12 weeks, and at 6 weeks were comparable to those of native articular cartilage. In histological examination, specimens showed neocartilage formation. There was no significant difference between ultrasound-treated and sham-treated groups. The mechanical stability of the neocartilage specimens increased with treatment time and reached values of native cartilage after 6 weeks in vivo. Low-intensity pulsed-ultrasound stimulation showed no stimulatory effect on tissue maturation. In contrast, ultrasound-treated specimens showed a reduced Col 2 expression at 1 week and were significantly less stiff compared to native cartilage at 6 and 12 weeks. An acceleration of the maturation of tissue-engineered neocartilage in a clinical setting by means of low-intensity pulsed ultrasound therefore appears rather unrealistic.

Cartilage↗
PubMed2004

Distinct roles of BMP receptors Type IA and IB in osteo-/chondrogenic differentiation in mesenchymal progenitors (C3H10T1/2).

The functional roles of BMP type IA and IB receptors mediating differentiation into the osteogenic and chondrogenic lineage were investigated in the mesenchymal progenitor line C3H10T1/2 in vitro. The capacity of type IA and IB BMP receptors was assessed by the forced expression of the wild-type (wtBMPR-IA or IB) and of the kinase-deficient, dominant-negative form (dnBMPR-IA or -IB) in parental C3H10T1/2 progenitors as well as in C3H10T1/2 progenitors which recombinantly express BMP2 (C3H10T1/2-BMP2) or GDF5 (C3H10T1/2-GDF5). Consistent with the higher endogenous expression rate of BMPR-IA in comparison with BMPR-IB, BMPR-IA plays the dominant role in BMP2-mediated osteo-/chondrogenic development. BMPR-IB moderately influences osteogenic and hardly chondrogenic development. BMPR-IB seems to be unable to efficiently activate downstream signaling pathways upon forced expression. However, a mutation conferring constitutive activity to the BMPR-IB receptor indicates that this receptor possesses the capacity to activate downstream signaling cascades. These results suggest that in mesenchymal progenitors C3H10T1/2 BMPR-IA is responsible for the initiation of the osteogenic as well as chondrogenic development and that BMPR-IA and -IB receptor pathways are well separated in this mesenchymal progenitor line and may not substitute each other. In addition this indicates that type IB and IA BMP receptors may transmit different signals during the specification and differentiation of mesenchymal lineages.

Animals↗
PubMed2004

Current strategies for cell delivery in cartilage and bone regeneration.

Several cell-based tissue-engineering therapies are emerging to regenerate damaged tissues. These strategies use autologous cells in combination with bioresorbable delivery materials. Major functions of a delivery scaffold are to provide initial mechanical stability, homogenous three-dimensional cell distribution, improved tissue differentiation, suitable handling and properties for delivery and fixation into patients. Delivery of cells can be achieved using injectable matrices, soft scaffolds, membranes, solid load-bearing scaffolds or immunoprotective macroencapsulation. Thus, to expand the clinical potential, next generation therapies will depend on smart delivery concepts that make use of the regenerative potential of stem cells, morphogenetic growth factors and biomimetic materials.

Animals↗
PubMed2004

Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems.

One of the milestones in tissue engineering has been the development of 3D scaffolds that guide cells to form functional tissue. Recently, mouldless manufacturing techniques, known as solid free-form fabrication (SFF), or rapid prototyping, have been successfully used to fabricate complex scaffolds. Similarly, to achieve simultaneous addition of cells during the scaffold fabrication, novel robotic assembly and automated 3D cell encapsulation techniques are being developed. As a result of these technologies, tissue-engineered constructs can be prepared that contain a controlled spatial distribution of cells and growth factors, as well as engineered gradients of scaffold materials with a predicted microstructure. Here, we review the application, advancement and future directions of SFF techniques in the design and creation of scaffolds for use in clinically driven tissue engineering.

Biocompatible Materials↗
PubMed2003

[Mesenchymal stem cells for tissue engineering of bone and cartilage].

The adult bone marrow contains mesenchymal stem cells. These cells show the capacity to undergo an extensive replication in vitro. In addition, mesenchymal stem cells have the potential to develop either in vitro or in vivo into mesenchymal tissues like bone and cartilage, which suggests these cells being an attractive source for tissue engineering approaches. Tissue engineering allows to isolate patient's own cells from tissue biopsies, to expand them, to combine them with carrier materials and growth factors, and to retransplant the constructs into the patient. Due to the prerequisite for tissue engineering to ensure a sufficient number of tissue-specific cells, much attention has been drawn to multipotential progenitor cells such as mesenchymal stem cells. In this article, important aspects of tissue engineering of bone and cartilage will be reviewed with emphasis on mesenchymal stem cells.

Bone Regeneration↗
PubMed2003

Immunomodulation of tissue-engineered transplants: in vivo bone generation from methylprednisolone-stimulated chondrocytes.

Subcutaneously implanted, in vitro engineered tissue is generally affected by the immune system of the host even in autogenous transplantation. The aim of this study was to investigate immunomodulation of subcutaneously implanted tissue-engineered cartilage transplants by intramuscular methylprednisolone application. Transplants consisted of auricular rabbit chondrocytes, polylactide-polyglycolide co-polymer fleeces and species-specific fibrin or agarose. The transplants were subcutaneously implanted in the ridge. Thereafter, animals were separated into two groups, one with and one without methylprednisolone treatment. The specimens were histologically investigated after 6 and 12 weeks. Fleece fiber degradation was complete after 12 weeks, and all transplants showed areas of calcification. The corticosteroid-treated group presented pronounced trabecular bone generation without fibrous tissue infiltration. The untreated group showed sporadic islets of calcification without coherent bone formation, and adjacent fibrous tissue had infiltrated the transplants. Native controls and corticoid-treated transplants did not exhibit bone generation or signs of fibrous tissue infiltration. This study found that immunomodulation by intramuscular methylprednisolone application protects tissue-engineered autogenous chondrocyte transplants from fibrous tissue infiltration and induces trabecular bone formation.

Animals↗
PubMed2003

Elastic cartilage engineering using novel scaffold architectures in combination with a biomimetic cell carrier.

Tissue engineering of an elastic cartilage graft that meets the criterion for both structural and functional integration into host tissue, as well as allowing for a clinically tolerable immune response, is a challenging endeavour. Conventional scaffold technologies have limitations in their ability to design and fabricate complex-shaped matrix architectures of structural and mechanical equivalence to elastic cartilage found in the body. We attempted to investigate the potential of conventionally isolated and passaged chondrocytes (2D environment) when seeded and cultured in combination with a biomimetic hydrogel in a mechanically stable and biomimetic composite matrix to form elastic cartilage within ectopic implantation sites. In vitro cultured scaffold/hydrogel/chondrocytes constructs showed islets of cartilage and mineralized tissue formation within the cell-seeded specimens in both pig and rabbit models. Specimens with no cells seeded showed only vascularized fibrous tissue ingrowth. These studies demonstrated the potential of such scaffold/hydrogel/cell constructs to support chondrogenesis in vivo. However, it also showed that even mechanically stable scaffolds do not allow regeneration of a large mass of structural and functional cartilage within a matrix architecture seeded with 2D passaged chondrocytes in combination with a cell biomimetic carrier. Hence, future experiments will be designed to evaluate an initial 3D culture of chondrocytes, effect on cell phenotype and their subsequent culture within biomimetic 3D scaffold/cell constructs.

Adult↗
PubMed2003

Making bone: implant insertion into tissue-engineered bone for maxillary sinus floor augmentation-a preliminary report.

Autologous, allogenic and alloplastic materials for bony reconstruction in the cranio-maxillofacial area have many drawbacks thus stimulating the on-going search for new (bio-)materials. Whereas cultivated skin and mucosa are already in clinical routine use in head and neck reconstruction, so far there has been no successful clinical application to the best of our knowledge of periosteum-derived, tissue-engineered bone for augmentation of the edentulous posterior maxilla. In a pilot study, augmentation of the posterior maxilla was carried out using a bone matrix derived from mandibular periosteal cells on a polymer fleece. This paper demonstrates fabrication of the matrix, clinical application, and the histological results in two patients. The results suggest that periosteum-derived osteoblasts on a suitable matrix form lamellar bone within 4 months which allows reliable implant insertion.

Alveolar Ridge Augmentation↗
PubMed2003

The arthroscopic implantation of autologous chondrocytes for the treatment of full-thickness cartilage defects of the knee joint.

Autologous chondrocyte implantation is an established option for the treatment of full-thickness cartilage defects of the knee. Open implantation has a high morbidity. On a resorbable polymer fleece, autologous chondrocytes can be implanted arthroscopically. Transosseous anchoring assures high initial stability of the implant. Tibial defects can be addressed. The arthroscopic technique for the implantation of autologous chondrocytes eliminates a substantial amount of the side effects known to occur after open autologous chondrocyte implantation procedures.

Arthroscopy↗