Dietmar Hutmacher

This paper reports on a 2-phase study of a novel membrane-scaffold graft construct, its ability to support periodontal ligament fibroblast (PDLF) and alveolar osteoblast (AO) growth in vitro, and its use for tissue engineering a PDL-AO interface in vivo. Human PDLFs were seeded onto perforated poly(epsilon-caprolactone) membranes (n=30) at 78,000 cells/cm2; human AOs were seeded on poly(epsilon-caprolactone) scaffolds (n=30) with fibrin glue at 625,000 cells/cm3. Cell attachment, morphology, viability, and metabolic activity were monitored for 3 weeks in vitro. Subsequently, cell-seeded membrane-scaffold constructs (experimental group, n=9) and nonseeded constructs (control group, n=4) assembled with fibrin glue were implanted subcutaneously into 7 athymic mice for 4 weeks. PDLFs formed confluent layers on membranes, whereas AOs produced mineralized matrices within scaffolds upon osteoinduction in vitro. Well-vascularized tissue formation was observed after implantation. Integration...

Tumour microenvironment greatly influences the development and metastasis of cancer progression. The development of three dimensional (3D) culture models which mimic that displayed in vivo can improve cancer biology studies and accelerate novel anticancer drug screening. Inspired by a systems biology approach, we have formed 3D in vitro bioengineered tumour angiogenesis microenvironments within a glycosaminoglycan-based hydrogel culture system. This microenvironment model can routinely recreate breast and prostate tumour vascularisation. The multiple cell types cultured within this model were less sensitive to chemotherapy when compared with two dimensional (2D) cultures, and displayed comparative tumour regression to that displayed in vivo. These features highlight the use of our in vitro culture model as a complementary testing platform in conjunction with animal models, addressing key reduction and replacement goals of the future. We anticipate that this biomimetic model will provide a platform for the in-depth analysis of cancer development and the discovery of novel therapeutic targets.

Bone metastasis is a frequent and life-threatening complication of breast cancer. The molecular mechanisms supporting the establishment of breast cancer cells in the skeleton are still not fully understood, which may be attributed to the lack of suitable models that interrogate interactions between human breast cancer cells and the bone microenvironment. Although it is well-known that integrins mediate adhesion of malignant cells to bone extracellular matrix, their role during bone colonization remains unclear. Here, the role of β1 integrins in bone colonization was investigated using tissue-engineered humanized in vitro and in vivo bone models. In vitro, bone-metastatic breast cancer cells with suppressed integrin β1 expression showed reduced attachment, spreading, and migration within human bone matrix compared to control cells. Cell proliferation in vitro was not affected by β1 integrin knockdown, yet tumor growth in vivo within humanized bone microenvironments was significantly ...

We have compared the effects of different sterilization techniques on the properties of Bombyx mori silk fibroin thin films with the view to subsequent use for corneal tissue engineering. The transparency, tensile properties, corneal epithelial cell attachment and degradation of the films were used to evaluate the suitability of certain sterilization techniques including gamma-irradiation (in air or nitrogen), steam treatment and immersion in aqueous ethanol. The investigations showed that gamma-irradiation, performed either in air or in a nitrogen atmosphere, did not significantly alter the properties of films. The films sterilized by gamma-irradiation or by immersion in ethanol had a transparency greater than 98% and tensile properties comparable to human cornea and amniotic membrane, the materials of choice in the reconstruction of ocular surface. Although steam-sterilization produced stronger, stiffer films, they were less transparent, and cell attachment was affected by the var...

Mesenchymal stem cells derived from human bone marrow (hBMSCs) and human adipose tissue (hAMSCs) represent a useful source of progenitor cells for cell therapy and tissue engineering. However, it is not clear what the similarities and differences between them are. Like hBMSCs, hAMSCs can differentiate into osteogenic, adipogenic, and chondrogenic cells. Whether MSCs derived from different tissue sources represent fundamentally similar or different cell types is not clear. Given the possible different sources of MSCs for cell therapy, a comprehensive comparison of the different MSCs would be very useful. Here, we compared the transcriptome profile of hAMCS and hBMSCs during directed differentiation into bone, cartilage, and fat. Our data revealed considerable similarities between bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (AMSCs). We uncovered an interesting bifurcation of pathways in both BMSCs and AMSCs, in which osteogenesis and adipogenesis appear to be link...

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces g...

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the

... Subha Narayan Rath a , Danny Cohn c & Professor Dietmar Werner Hutmacher PhD (NUS)MBA (Henley) a b * pages 209-219. ... 200414. Heywood, HK 2004. Cellular utilization determines viability and matrix distribution profiles in chondrocyte-seeded alginate constructs. ...

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ε-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

... 1 DIETMAR W. HUTMACHER, Ph.D., MBA,1,2 JAN-THORSTEN SCHANTZ, MD,1 CHIN SENG NG ... Teoh, SH, Tang, ZG, and Ramakrishna, S. Development of thin elastomeric composite membranes for biomed-ical applications. ... [CrossRef] 3. Chen-Huan Lin, Shan-hui Hsu, Jang ...

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.

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.

A novel strategy is reported to produce biodegradable microfiber-scaffolds layered with high densities of microparticles encapsulating a model protein. Direct electrospraying on highly porous melt electrospun scaffolds provides a reproducible scaffold coating throughout the entire architecture. The burst release of protein is significantly reduced due to the immobilization of microparticles on the surface of the scaffold and release mechanisms are dependent on protein-polymer interactions. The composite scaffolds have a positive biological effect in contact with precursor osteoblast cells up to 18 days in culture. The scaffold design achieved with the techniques presented here endorses these new composite scaffolds as promising templates for growth factor delivery.