TisXell Publications

Author information

1
Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, 4059 Kelvin Grove, Brisbane, Australia.
2
Department of Orthopaedics, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands.
3
Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, 4059 Kelvin Grove, Brisbane, Australia; Institute for Advanced Study, Technical University Munich, Lichtenbergstrasse 2a, 85748 Garching, Munich, Germany. Electronic address: dietmar.hutmacher@qut.edu.au.

Abstract

Adipose tissue engineering offers a promising alternative to the current breast reconstruction options. Here we investigated patient-specific breast scaffolds fabricated from poly(d,l)-lactide polymer with pore sizes>1 mm for their potential in long-term sustained regeneration of high volume adipose tissue. An optimised scaffold geometry was modelled in silico via a laser scanning data set from a patient who underwent breast reconstruction surgery. After the design process scaffolds were fabricated using an additive manufacturing technology termed fused deposition modelling. Breast-shaped scaffolds were seeded with human umbilical cord perivascular cells and cultured under static conditions for 4 weeks and subsequently 2 weeks in a biaxial rotating bioreactor. These in vitro engineered constructs were then seeded with human umbilical vein endothelial cells and implanted subcutaneously into athymic nude rats for 24 weeks. Angiogenesis and adipose tissue formation were observed throughout all constructs at all timepoints. The percentage of adipose tissue compared to overall tissue area increased from 37.17% to 62.30% between week 5 and week 15 (p<0.01), and increased to 81.2% at week 24 (p<0.01), while the seeded endothelial cells self-organised to form a functional capillary network. The presented approach of fabricating customised scaffolds using 3D scans represents a facile approach towards engineering clinically relevant volumes of adipose tissue for breast reconstruction.

Author information

1

Chair Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia. Dietmar.Hutmacher@qut.edu.au

Abstract

The complex relationship between the hydrodynamic environment and surrounding tissues directly impacts on the design and production of clinically useful grafts and implants. Tissue engineers have generally seen bioreactors as ‘black boxes’ within which tissue engineering constructs (TECs) are cultured. It is accepted that a more detailed description of fluid mechanics and nutrient transport within process equipment can be achieved by using computational fluid dynamics (CFD) technology. This review discusses applications of CFD for tissue engineering-related bioreactors — fluid flow processes have direct implications on cellular responses such as attachment, migration and proliferation. We conclude that CFD should be seen as an invaluable tool for analyzing and visualizing the impact of fluidic forces and stresses on cells and TECs.

Author information

1Department of Mechanical Engineering, National University of Singapore.

Abstract

Today, tissue engineers are attempting to engineer virtually every human tissue. Potential tissue-engineered products include cartilage, bone, heart valves, nerves, muscle, bladder, liver, etc. Tissue engineering techniques generally require the use of a porous scaffold, which serves as a three-dimensional template for initial cell attachment and subsequent tissue formation both in vitro and in vivo. The scaffold provides the necessary support for cells to attach, proliferate, and maintain their differentiated function. Its architecture defines the ultimate shape of the new grown soft or hard tissue. In the early days of tissue engineering, clinically established materials such as collagen and polyglycolide were primarily considered as the material of choice for scaffolds. The challenge for more advanced scaffold systems is to arrange cells/tissue in an appropriate 3D configuration and present molecular signals in an appropriate spatial and temporal fashion so that the individual cells will grow and form the desired tissue structures–and do so in a way that can be carried out reproducibly, economically, and on a large scale. This paper is not intended to provide a general review of tissue engineering, but specifically concentrate on the design and processing of synthetic polymeric scaffolds. The material properties and design requirements are discussed. An overview of the various fabrication techniques of scaffolds is presented, beginning with the basic and conventional techniques to the more recent, novel methods that combine both scaffold design and fabrication capabilities.

PMID:
11334185
DOI:
10.1163/156856201744489

Hutmacher DW1Schantz TZein INg KWTeoh SHTan KC.

Author information

1
Laboratory for Biomedical Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. mpedwh@nus.edu.sq

Abstract

A number of different processing techniques have been developed to design and fabricate three-dimensional (3D) scaffolds for tissue-engineering applications. The imperfection of the current techniques has encouraged the use of a rapid prototyping technology known as fused deposition modeling (FDM). Our results show that FDM allows the design and fabrication of highly reproducible bioresorbable 3D scaffolds with a fully interconnected pore network. The mechanical properties and in vitro biocompatibility of polycaprolactone scaffolds with a porosity of 61 +/- 1% and two matrix architectures were studied. The honeycomb-like pores had a size falling within the range of 360 x 430 x 620 microm. The scaffolds with a 0/60/120 degrees lay-down pattern had a compressive stiffness and a 1% offset yield strength in air of 41.9 +/- 3.5 and 3.1 +/- 0.1 MPa, respectively, and a compressive stiffness and a 1% offset yield strength in simulated physiological conditions (a saline solution at 37 degrees C) of 29.4 +/- 4.0 and 2.3 +/- 0.2 MPa, respectively. In comparison, the scaffolds with a 0/72/144/36/108 degrees lay-down pattern had a compressive stiffness and a 1% offset yield strength in air of 20.2 +/- 1.7 and 2.4 +/- 0.1 MPa, respectively, and a compressive stiffness and a 1% offset yield strength in simulated physiological conditions (a saline solution at 37 degrees C) of 21.5 +/- 2.9 and 2.0 +/- 0.2 MPa, respectively. Statistical analysis confirmed that the five-angle scaffolds had significantly lower stiffness and 1% offset yield strengths under compression loading than those with a three-angle pattern under both testing conditions (p < or = 0.05). The obtained stress-strain curves for both scaffold architectures demonstrate the typical behavior of a honeycomb structure undergoing deformation. In vitro studies were conducted with primary human fibroblasts and periosteal cells. Light, environmental scanning electron, and confocal laser microscopy as well as immunohistochemistry showed cell proliferation and extracellular matrix production on the polycaprolactone surface in the 1st culturing week. Over a period of 3-4 weeks in a culture, the fully interconnected scaffold architecture was completely 3D-filled by cellular tissue. Our cell culture study shows that fibroblasts and osteoblast-like cells can proliferate, differentiate, and produce a cellular tissue in an entirely interconnected 3D polycaprolactone matrix.

Lam CX1Hutmacher DWSchantz JTWoodruff MATeoh SH.

Author information

1
Division of Bioengineering, National University of Singapore, Blk E3A #04-15, 7 Engineering Dr 1, Singapore 117574.

Abstract

The use of polycaprolactone (PCL) as a biomaterial, especially in the fields of drug delivery and tissue engineering, has enjoyed significant growth. Understanding how such a device or scaffold eventually degrades in vivo is paramount as the defect site regenerates and remodels. Degradation studies of three-dimensional PCL and PCL-based composite scaffolds were conducted in vitro (in phosphate buffered saline) and in vivo (rabbit model). Results up to 6 months are reported. All samples recorded virtually no molecular weight changes after 6 months, with a maximum mass loss of only about 7% from the PCL-composite scaffolds degraded in vivo, and a minimum of 1% from PCL scaffolds. Overall, crystallinity increased slightly because of the effects of polymer recrystallization. This was also a contributory factor for the observed stiffness increment in some of the samples, while only the PCL-composite scaffold registered a decrease. Histological examination of the in vivo samples revealed good biocompatibility, with no adverse host tissue reactions up to 6 months. Preliminary results of medical-grade PCL scaffolds, which were implanted for 2 years in a critical-sized rabbit calvarial defect site, are also reported here and support our scaffold design goal for gradual and late molecular weight decreases combined with excellent long-term biocompatibility and bone regeneration.

PMID:
18646204
DOI:
10.1002/jbm.a.32052

Leferink AM1Chng YC2van Blitterswijk CA1Moroni L1.

Author information

1
Department of Tissue Regeneration, MIRA Institute, University of Twente , Enschede , Netherlands ; Department of Complex Tissue Regeneration, Faculty of Health, Medicine and Life Sciences, Maastricht University , Maastricht , Netherlands.
2
Quintech Life Sciences Pte Ltd. , Singapore , Singapore.

Abstract

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactorafter static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

KEYWORDS: biaxial rotating bioreactor; bone marrow stromal cells; cellular distribution; perfusion-flow bioreactor; scaffolds

PMID:
26557644
PMCID:
PMC4617101
DOI:
10.3389/fbioe.2015.00169

Author information

1
Laboratoire Matière et Systèmes Complexes (MSC), UMR 7057 CNRS & University Paris Diderot, Paris F-75205 Cedex 13, France. Electronic address: nathalie.luciani@univ-paris-diderot.fr.
2
Laboratoire Matière et Systèmes Complexes (MSC), UMR 7057 CNRS & University Paris Diderot, Paris F-75205 Cedex 13, France.
3
Laboratoire de recherche vasculaire translationnelle, INSERM UMR 1148 & University Paris Diderot, Paris, France.
4
INSERM, U791, LIOAD, Nantes F-44042, France.

Abstract

Tissue engineering strategies, such as cellularized scaffolds approaches, have been explored for cartilage replacement. The challenge, however, remains to produce a cartilaginous tissue incorporating functional chondrocytes and being large and thick enough to be compatible with the replacement of articular defects. Here, we achieved unprecedented cartilage tissue production into a porous polysaccharide scaffold by combining of efficient magnetic condensation of mesenchymal stem cells, and dynamic maturation in a bioreactor. In optimal conditions, all the hallmarks of chondrogenesis were enhanced with a 50-fold increase in collagen II expression compared to negative control, an overexpression of aggrecan and collagen XI, and a very low expression of collagen I and RUNX2. Histological staining showed a large number of cellular aggregates, as well as an increased proteoglycan synthesis by chondrocytes. Interestingly, electron microscopy showed larger chondrocytes and a more abundant extracellular matrix. In addition, the periodicity of the neosynthesized collagen fibers matched that of collagen II. These results represent a major step forward in replacement tissue for cartilage defects.

STATEMENT OF SIGNIFICANCE:

A combination of several innovative technologies (magnetic cell seeding, polysaccharide porous scaffolds, and dynamic maturation in bioreactor) enabled unprecedented successful chondrogenesis within scaffolds. 

KEYWORDS: Bioreactor; Cartilage defect; Chondrogenesis; Magnetic mesenchymal stem cells; Tissue engineering

Rai B1Teoh SHHo KHHutmacher DWCao TChen FYacob K.

Author information

1
Department of bioengineering, Faculty of Dentistry, National University of Singapore, Singapore, Singapore. g0201957@nus.edu.sg

Abstract

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblastsseeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.

Author information

1
Tissue Engineering Laboratory E3-05-04, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore. g0301488@nus.edu.sg

Abstract

The problem of donor scarcity has led to the recent development of tissue engineering technologies, which aim to create implantable tissue equivalents for clinical transplantation. These replacement tissues are being realised through the use of biodegradable polymer scaffolds; temporary/permanent substrates, which facilitate cell attachment, proliferation, retention and differentiated tissue function. To optimise gas transfer and nutrient delivery, as well as to mimic the fluid dynamic environment present within the body, a dynamic system might be chosen. Experiments have shown that dynamic systems enhance tissue growth, with the aid of scaffolds, as compared to static culture systems. Very often, tissue growth within scaffolds is only seen to occur at the periphery. The present study utilises the Computational Fluid Dynamics package FLUENT, to provide a better understanding of the flow phenomena in scaffolds, within our novel bioreactor system. The uni-axial and bi-axial rotational schemes are studied and compared, based on a vessel rotating speed of 35 rpm. The wall shear stresses within and without the constructs are also studied. Findings show that bi-axial rotation of the vessel results in manifold increases of fluid velocity within the constructs, relative to uni-axial rotation about the X- and Z-axes, respectively.

Zhang ZY1Teoh SHChong MSLee ESTan LGMattar CNFisk NMChoolani MChan J.

Author information

1
Mechanical Engineering, Faculty of Engineering, National University of Singapore, Singapore.

Abstract

Tissue-engineered bone grafts (TEBG) require highly osteogenic cell sources for use in fracture repair applications. Compared to other sources of mesenchymal stem cells (MSC), human fetal MSC (hfMSC) have recently been shown to be more proliferative and osteogenic. We studied the functional performance of hfMSC-mediated TEBG in 7 mm rat femoral critical-sized bone defects (CSD). Dynamically-cultured and osteogenically-primed hfMSC seeded onto macroporous poly-epsilon-caprolactone tri-calcium phosphate scaffolds were transplanted into CSDs. After 12 weeks, hfMSC-mediated TEBG induced 2.1x more new bone formation (43.3+/-10.5 vs. 21.0+/-7.4 mm(3), p<0.05), with greater compact and woven bone, and a 9.8x increase in stiffness (3.9+/-1.7 vs. 0.4+/-0.3 mNm/degree, p<0.05) compared to acellular scaffolds, such that only animals transplanted with TEBG underwent full fracture repair of the CSD. Although hfMSC survived for <4 weeks, by 4 weeks they were associated with a 3.9x larger vasculature network in the defect area (35.2+/-11.1 vs. 6.5+/-3.6 mm(3)p<0.05), suggesting an important role for hfMSC in the promotion of neo-vasculogenesis. We speculate that hfMSC-mediated healing of the CSD by stimulating neo-vascularization through as yet undetermined mechanisms. This proof-of-principle study demonstrates the utility of primitive MSC for bone regeneration, and may be of relevance to vascularization in other areas of regenerative medicine.

Zhang ZY1Teoh SHChong MSSchantz JTFisk NMChoolani MAChan J.

Author information

1
National University of Singapore, Singapore.

Abstract

Mesenchymal stem cells (MSCs) from human adult bone marrow (haMSCs) represent a promising source for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. Alternative postnatal, perinatal, and fetal sources of MSCs appear to have different osteogenic capacities, but have not been systematically compared with haMSCs. We investigated the proliferative and osteogenic potential of MSCs from human fetal bone marrow (hfMSCs), human umbilical cord (hUCMSCs), and human adult adipose tissue (hATMSCs), and haMSCs, both in monolayer cultures and after loading into three-dimensional polycaprolactone-tricalcium-phosphate scaffolds.Although all MSCs had comparable immunophenotypes, only hfMSCs and hUCMSCs were positive for the embryonic pluripotency markers Oct-4 and Nanog. hfMSCs expressed the lowest HLA-I level (55% versus 95%-99%) and the highest Stro-1 level (51% versus 10%-27%), and had the greatest colony-forming unit-fibroblast capacity (1.6x-2.0x; p < .01) and fastest doubling time (32 versus 54-111 hours; p < .01). hfMSCs had the greatest osteogenic capacity, as assessed by von-Kossa staining, alkaline phosphatase activity (5.1x-12.4x; p < .01), calcium deposition (1.6x-2.7x in monolayer and 1.6x-5.0x in scaffold culture; p < .01), calcium visualized on micro-computed tomography (3.9×17.6x; p < .01) and scanning electron microscopy, and osteogenic gene induction. Two months after implantation of cellular scaffolds in immunodeficient mice, hfMSCs resulted in the most robust mineralization (1.8x-13.3x; p < .01).The ontological and anatomical origins of MSCs have profound influences on the proliferative and osteogenic capacity of MSCs. hfMSCs had the most proliferative and osteogenic capacity of the MSC sources, as well as being the least immunogenic, suggesting they are superior candidates for bone tissue engineering.

Author information

1
Graduate Program in Bioengineering (GPBE), National University of Singapore, Singapore.

Abstract

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity. and maintained high cellular viability in the core, which was 2000 microm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression , calcium deposition  and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice  compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.

PMID:19223070
DOI:10.1016/j.biomaterials.2009.01.028

Zhang ZY1Teoh SHHui JHFisk NMChoolani MChan JK.

Author information

1
Mechanical Engineering, Faculty of Engineering, National University of Singapore, Singapore.

Abstract

Mesenchymal stem cells (MSCs) have become one of the most promising cell sources for bone tissue engineering (BTE) applications. In this review, we first highlight recent progress in the understanding of MSC biology, their in vivo niche, multi-faceted contribution to fracture healing and bone re-modelling, and their role in BTE. A literature review from clinicaltrials.gov and Pubmed on clinical usage of MSC for both orthopedic and non-orthopedic indications suggests that translational use of MSC for BTE indications is likely to bear fruit in the ensuing decade. Last, we disscuss the profound influence of ontological and antomical origins of MSC on their proliferation and osteogenesis and demonstrated human fetal MSC (hfMSC) as a superior cellular candidate for off-the-shelf BTE applications. This relates to their superior proliferation capacity, more robust osteogenic potential and lower immunogenecity, as compared to MSC from perinatal and postnatal sources. Furthermore, we discuss our experience in developing a hfMSC based BTE strategy with the integrated use of bioreactor-based dynamic priming within macroporous scaffolds, now ready for evaluation in clinical trials. In conclusion, hfMSC is likely the most promising cell source for allogeneic based BTE application, with proven advantages compared to other MSC based ones.