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Biomedical subjects

Dietmar Werner Hutmacher

Publications and source records attributed to Dietmar Werner Hutmacher.

12 recordsLinked to original sources
PubMed2006

Absolute quantification of gene expression in biomaterials research using real-time PCR.

One major measurement of tissue-engineered constructs efficacy and performance is determining expression levels of genes of interest at the molecular level. This measurement is commonly carried out with reverse transcription-polymerase chain reaction (RT-PCR). In this study, we described a novel method in achieving absolute quantification of gene expression using real-time PCR (aqPCR). This novel method did not require molecular cloning steps to prepare the standards for quantification comparison. Standards were linear double-stranded DNA molecules instead of the typical gene-in-plasmid format. aqPCR could also be used to give relative quantification comparisons between samples simply by dividing the copy numbers readings of the gene of interest with that of the normalization gene. RNA was extracted from monolayer and from polycaprolactone scaffold cultures and assayed for beta-actin and osteocalcin genes. We compared our aqPCR method with end-point PCR since end-point PCR is still a common means of measuring gene expression in the biomaterials field. This study showed that aqPCR was a better method to quantify gene expression than end-point PCR. With our described linear DNA standards method, we were able to obtain not only relative quantification of osteocalcin and beta-actin expression level but also actual copy numbers of osteocalcin and beta-actin for the monolayer culture and to be 1.34 x 10(4) and 1.45 x 10(7) copies, respectively and for the scaffold cultures to be 772 and 2.83 x 10(5) copies, respectively per starting total RNA mass of 10 ng. The standards curves made from these linear DNA standards showed good linearity (R(2)=0.9964 and 0.9902 for osteocalcin and beta-actin standards graphs), ranged from 10 to 10(9) copies and of comparable accuracy to current absolute quantification real-time PCR methods (which used plasmid standards obtained through molecular cloning methods). Our method might be a viable and more user-friendly alternative to current absolute quantification real-time PCR protocols.

Biocompatible Materials↗
PubMed2006

Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices.

In order to alleviate their extensive contraction, human fibroblast sheets were cultured in combination with three-dimensional matrices (knitted poly(lactic-co-glycolic acid) (PLGA) mesh and collagen-hyaluronic acid (CHA) sponge) to form contiguous dermal constructs for tissue engineering a bilayered skin equivalent. The resulting constructs were viable, and supported the development of bilayered skin equivalents which did not contract over the 4-week culture period. When implanted into full-thickness wounds in nude rats, cultured skin equivalents based on PLGA meshes registered a take rate of 100% and showed an extent of wound contraction that was statistically similar to autografts, while wounds grafted with PLGA meshes without cell sheets contracted more than autografts. On the other hand, skin equivalents based on CHA sponges were all sloughed off within 2 weeks of transplantation. In all cell sheet-incorporated specimens, cells from the constructs infiltrated and produced extracellular matrix within the neo-dermis, shown by positive human leukocyte antigen and collagen I expression. This technique offers an alternative approach for scaffold-based tissue engineering to produce mechanically stable grafts with matured neo-tissue.

Animals↗
PubMed2006

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model.

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced--stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced--induced for 2 weeks before seeding into scaffolds; and (3) uninduced--cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.

Adipose Tissue↗
PubMed2006

Cranioplasty after trephination using a novel biodegradable burr hole cover: technical case report.

OBJECTIVE AND IMPORTANCE: We have developed novel biodegradable polymer implants by using the rapid prototyping technology fused deposition modeling. Early results of a clinical pilot study for cranioplasty are presented. CLINICAL PRESENTATION: Five patients with the diagnosis of chronic subdural hematoma were included in the study. After trephination and evacuation of the subdural hematoma, burr holes (diameter, 14 mm) were closed using a biodegradable implant made of polycaprolactone. Implants were computer designed with an upper rim diameter of 16 mm and a 14 mm body diameter with a fully interconnected, honeycomb-like architecture of 400 to 600 microm in pore size. INTERVENTION: Postoperative computed tomographic scans indicated that the plugs were stably anchored in the osseous host environment with no fluid collection detectable. The postoperative course was uneventful, and patients were discharged after 5 days. Follow-up scans after 3, 6, and 12 months showed that the implants were well integrated in the surrounding calvarial bone with new bone filling the porous space. CONCLUSION: These novel polymer scaffolds made of the slow-degrading material polycaprolactone represent a suitable implant for closure of post-trephination defects.

Absorbable Implants↗
PubMed2005

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering.

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Actins↗
PubMed2005

Osteogenic differentiation of mesenchymal progenitor cells in computer designed fibrin-polymer-ceramic scaffolds manufactured by fused deposition modeling.

Biomimetic scaffolds offer great potentials in the development of bone analogs for tissue engineering. The studies presented in this paper focus specifically on the osteogenic potential of the novel PCL/CaP matrices and its degradation behavior. Biodegradable Polymer-ceramic Scaffolds were fabricated using the solid free form fabrication technology: Fused Deposition Modeling (FDM). The scaffold architecture was characterized by a honeycomb-like design and a complete interconnectivity of the pores. Human mesenchymal stem cells (MSCs) were seeded together with fibrin glue into PCL/CaP scaffolds and cultured in vitro for periods of up to eight weeks. Cellular adhesion, proliferation and osteogenic differentiation were assessed in these constructs using a range of histological and microscopic techniques. In additional experiments, degradation was assessed by measuring mass loss, diameter change, molecular weight change and by scanning electron micrographs. MSCs were able to adhere, migrate, and differentiate along the osteogenic lineage with in these scaffolds. The PCL/CaP scaffolds showed up to 27 fold increased degradation of compared to PCL scaffolds.

Biodegradation, Environmental↗
PubMed2004

Degradation and cell culture studies on block copolymers prepared by ring opening polymerization of epsilon-caprolactone in the presence of poly(ethylene glycol).

Poly(epsilon-caprolactone) (PCL) and its block copolymers with poly(ethylene glycol) (PEG) were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of ethylene glycol or PEG, using zinc metal as catalyst. The resulting polymers were characterized by various analytical techniques such as (1)H NMR, SEC, DSC, IR, X-ray, ESEM, and CZE. PCL/PEG copolymers with long PCL chains presented the same crystalline structure as PCL homopolymer, whereas PEG-bearing short PCL blocks retained the crystalline structure of PEG and exhibited an amphiphilic behavior in aqueous solutions. Degradation of PCL and PCL/PEG diblock and triblock copolymers was realized in a 0.13 M, pH 7.4 phosphate buffer at 37 degrees C. The results indicated that the copolymers exhibited higher hydrophilicity and degradability compared with the PCL homopolymer. Large amounts of PEG were released from the bulk after 60 weeks' degradation. In vitro cell culture studies were conducted on scaffolds manufactured via solid free form fabrication by using primary human and rat bone marrow derived stromal cells (hMSC, rMSC). Light, scanning electron, and confocal laser microscopy, as well as immunocytochemistry, showed cell attachment, proliferation, and extracellular matrix production on the surface, as well as inside the scaffold architecture. Copolymers showed better performance in the cell culture studies than the PCL homopolymer.

Animals↗
PubMed2003

Repair of calvarial defects with customized tissue-engineered bone grafts I. Evaluation of osteogenesis in a three-dimensional culture system.

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Animals↗
PubMed2003

Repair of calvarial defects with customised tissue-engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo.

We have demonstrated in Part I of this study [see Schantz, J.-T., et al., Tissue Eng. 2003;9(Suppl. 1): S-113-S-126; this issue] that bone marrow-derived progenitor cells and calvarial osteoblasts could be successfully directed into the osteogenic lineage and cultured in three-dimensional (3-D) polycaprolactone (PCL) scaffolds. The objective of the second part of the study was to evaluate and to compare tissue engineered cell-polymer constructs using calvarial osteoblasts (group I) and mesenchymal progenitor cells (MPCs; group II) for the reconstruction of critical-size and three-dimensionally complex cranial defects. In 30 New Zealand White rabbits, bilateral parietal critical-size defects were created. On the basis of computed tomography scans, customized PCL scaffolds with precisely controlled microarchitecture were fabricated, using a rapid prototyping technology. Bone marrow-derived progenitor cells and osteoblasts were isolated and expanded in culture. Osteoblasts (group I) and mesenchymal progenitor cells (group II) were seeded in combination with a fibrin glue suspension into 40 PCL scaffolds. After incubating for 3 days in static culture, the PCL scaffold-cell constructs as well as nonseeded PCL scaffolds (control group) were implanted into 15-mm-diameter calvarial defects. Reconstruction of the cranium and bone formation were evaluated after 3 months. In vivo results indicated osseous tissue integration within the implant and functionally stable restoration of the calvarium. Islands of early bone formation could be observed in X-ray radiographs and in histological sections. Implants showed a high cell:ECM ratio and a dense vascular network. Mechanical testing of the reconstructed area revealed partial integration with the surrounding corticocancellous calvarial bone. The amount (area) of calcification, measured by clinical computed tomography, indicated that cell-seeded constructs measured about 60% more than unrepaired or unseeded scaffolds. Mechanical investigations revealed that stiffness reached 52 +/- 29 and 44 +/- 16 MPa for MPC- and osteoblast-seeded scaffolds, respectively. The yield strength for the push-out tests was 180 +/- 36 N for normal calvarial bone, 90 +/- 1 N for unrepaired site, and 106 +/- 10 N for unseeded constructs, which is about 60% of normal bone strength. MPC- and osteoblast-seeded scaffolds indicated a yield strength of 149 +/- 15 and 164 +/- 42 N, respectively, which is about 85-90% of normal bone. This study demonstrated that customized biodegradable polymeric implants may be used to deliver osteogenic cells and enhance bone formation within critically-sized defects in vivo. The use of rapid prototyping technology to produce scaffolds with controlled external geometry and microarchitecture offers new possibilities in the functional and aesthetic reconstruction of complex craniofacial defects.

Animals↗
PubMed2002

Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology.

Due to their osteogenic germination potential, periosteum-derived osteoprogenitor cells are a potential source for tissue engineering a bone graft that could be used to regenerate skeletal defects. In this study we evaluated if ectopic bone formation could be induced by a construct made of human periosteal cells and a novel scaffold architecture whose mechanical properties are in the range of cancellous bone. Biopsies from human calvarial periosteum were harvested and cells were isolated from the inner cambial layer. Fifty thousand periosteal cells were seeded into the scaffolds measuring 6 x 6 x 2 mm. The cell-scaffold constructs were cultured for a period of 3 weeks prior to implantation into balb C nude mice. Mice were sacrificed and implants were analyzed 6 and 17 weeks postoperatively. Immunohistochemical analysis confirmed the osteoblastic phenotype of the seeded cells. Formation of focal adhesions and stress fibers could be observed in both scaffold architectures. Three-dimensional cell proliferation was observed after 2 weeks of culturing with centripetal growth pattern inside the pore network. The deposition of calcified extracellular matrix was observed after 3 weeks of culturing. In vivo, endochondral bone formation with osteoid production was detectable via von Kossa and Osteocalcin staining after 6 and 17 weeks. Histology and SEM revealed that the entire scaffold/bone grafts were penetrated by a vascular network. This study showed the potential of bone tissue engineering by using human periosteal cells in combination with a novel scaffold technology.

Animals↗
PubMedDate not supplied

Evaluation of a tissue-engineered membrane-cell construct for guided bone regeneration.

PURPOSE: Currently, a number of bioresorbable and biodegradable membranes used for guided bone regeneration lead to incomplete tissue regeneration. Poor mechanical properties, short degradation time, and the lack of integrated biologic components result in the inability to create and maintain an appropriate environment and to actively support tissue remodeling. In the present study, the osteogenic potential of human calvarial periosteal cells in combination with ultrathin polycaprolactone (pc2) membranes of a slow biodegradation rate was investigated. MATERIALS AND METHODS: In vitro and in vivo analyses of the tissue-engineered constructs were conducted using imaging techniques, immunohistochemistry, and histology. Two types of membranes were investigated. Group 1 consisted of a plain membrane, and in group 2 membranes were treated with sodium hydroxide. RESULTS: In vitro results showed that osteoblast-like cells attached and proliferated on the membranes with the formation of extracellular matrix. Sodium hydroxide-treated membranes showed enhanced cell attachment and proliferation kinetics, resulting in a dense cellular layer after 2 weeks in culture. In vivo mineralized tissue formation in association with vascularization was observed. Extracellular matrix calcification with nodule formation was detected via histology as well as scanning electron microscopy. DISCUSSION: PCL membranes support the attachment, growth, and osteogenic differentiation of human primary osteoblast-like cells. Sodium hydroxide-treated membranes demonstrated increased cell attachment resulting from increased hydrophilicity. CONCLUSION: These findings have potential application in the development of a new generation of osteoconductive membranes.

Absorbable Implants↗