Design of custom-shaped vascularized tissues using microtissue spheroids as minimal building units

Tissue engineering strategies are gathering clinical momentum in regenerative medicine and are expected to provide excellent opportunities for therapy for difficult-to-treat human pathologies. Being aware of the requirement to produce larger artificial tissue implants for clinical applications, we used microtissues, produced using gravity-enforced self-assembly of monodispersed primary cells, as minimal tissue units to generate scaffold-free vascularized artificial macrotissues in custom-shaped agarose molds. Mouse myoblast, pig and human articular-derived chondrocytes, and human myofibroblast (HMF)-composed microtissues (microm3 scale) were amalgamated to form coherent macrotissue patches (mm3 scale) of a desired shape. Macrotissues, assembled from the human umbilical vein endothelial cell (HUVEC)-coated HMF microtissues, developed a vascular system, which functionally connected to the chicken embryo's vasculature after implantation. The design of scaffold-free vascularized macrotissues is a first step toward the scale-up and production of artificial tissue implants for future tissue engineering initiatives.

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.

Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction

Tissue engineering represents an attractive approach for the treatment of congestive heart failure. The influence of the differentiation of myogenic graft for functional recovery is not defined. We engineered a biodegradable skeletal muscle graft (ESMG) tissue and investigated its functional effect after implantation on the epicardium of an infarcted heart segment. ESMGs were synthesized by mixing collagen (2 mg/mL), Matrigel (2 mg/mL), and rat skeletal muscle cells (10(6)). Qualitative and quantitative aspects of ESMGs were optimized. Two weeks following coronary ligation, the animals were randomized in three groups: ESMG glued to the epicardial surface with fibrin (ESMG, n = 7), fibrin alone (fibrin, n = 5), or sham operation (sham, n = 4). Echocardiography, histology, and immunostaining were performed 4 weeks later. A cohesive three-dimensional tissular structure formed in vitro within 1 week. Myoblasts differentiated into randomly oriented myotubes. Four weeks postimplantation, ESMGs were vascularized and invaded by granulation tissue. Mean fractional shortening (FS) was, however, significantly increased in the ESMG group as compared with preimplantation values (42 +/- 6 vs. 33 +/- 5%, P < 0.05) and reached the values of controlled noninfarcted animals (control, n = 5; 45 +/- 3%; not significant). Pre- and postimplantation FS did not change over these 4 weeks in the sham group and the fibrin-treated animals. This study showed that it is possible to improve systolic heart function following myocardial infarction through implantation of differentiated muscle fibers seeded on a gel-type scaffold despite a low rate of survival.

Stem cells as tools in regenerative therapy for retinal degeneration

OBJECTIVE: To describe the use of stem cells (SCs) for regeneration of retinal degenerations. Regenerative medicine intends to provide therapies for severe injuries or chronic diseases where endogenous repair does not sufficiently restore the tissue. Pluripotent SCs, with their capacity to give rise to specialized cells, are the most promising candidates for clinical application. Despite encouraging results, a combination with up-to-date tissue engineering might be critical for ultimate success. DESIGN: The focus is on the use of SCs for regeneration of retinal degenerations. Cell populations include embryonic, neural, and bone marrow-derived SCs, and engineered grafts will also be described. RESULTS: Experimental approaches have successfully replaced damaged photoreceptors and retinal pigment epithelium using endogenous and exogenous SCs. CONCLUSIONS: Stem cells have the potential to significantly impact retinal regeneration. A combination with bioengineering may bear even greater promise. However, ethical and scientific issues have yet to be solved.

Surgical suturing of articular cartilage induces osteoarthritis-like changes

INTRODUCTION: In clinical tissue-engineering-based approaches to articular cartilage repair, various types of flap are frequently used to retain an implanted construct within the defect, and they are usually affixed by suturing. We hypothesize that the suturing of articular cartilage is associated with a loss of chondrocytes from, and osteoarthritis-like changes within, the perisutural area. MATERIALS AND METHODS: We established a large, partial-thickness defect model in the femoral groove of adult goats. The defects were filled with bovine fibrinogen to support a devitalized flap of autologous synovial tissue, which was sutured to the surrounding articular cartilage with single, interrupted stitches. The perisutural and control regions were analyzed histologically, histochemically and histomorphometrically shortly after surgery and 3 weeks later. RESULTS: Compared to control regions, chondrocytes were lost from the perisutural area even during the first few hours of surgery. During the ensuing 3 weeks, the numerical density of cells in the perisutural area decreased significantly. The cell losses were associated with a loss of proteoglycans from the extracellular matrix. Shortly after surgery, fissures were observed within the walls of the suture channels. By the third week, their surface density had increased significantly and they were filled with avascular mesenchymal tissue. CONCLUSIONS: The suturing of articular cartilage induces severe local damage, which is progressive and reminiscent of that associated with the early stages of osteoarthritis. This damage could be most readily circumvented by adopting an alternative mode of flap affixation, such as gluing with a biological adhesive.

Wear particles and surface topographies are modulators of osteoclastogenesis in vitro

Prosthetic and osteosynthetic implants from metal alloys will be indispensable in orthopedic surgery, as long as tissue engineering and biodegradable bone substitutes do not lead to products that will be applied in clinical routine for the repair of bone, cartilage, and joint defects. Therefore, the elucidation of the interactions between the periprosthetic tissues and the implant remains of clinical relevance and several factors are known to affect the longevity of implants. Within this study, the effects of metal particles and surface topography on the recruitment of osteoclasts was investigated in vitro in a coculture of osteoblasts and bone marrow cells. The cells were grown in the presence of particles of different sizes and chemical composition or on metal discs with polished or sandblasted surfaces, respectively. At the end of the culture, newly formed osteoclasts were counted. Osteoclastogenesis was reduced when particles were added directly to the coculture. The effect depended on the size of the particles, small particles exerting stronger effects than larger ones. The chemical composition of the particles, however, did not affect the development of osteoclasts. In cocultures grown on sandblasted surfaces, osteoclasts developed at higher rates than they did in cultures on polished surfaces. The data demonstrate that wear particles and implant surfaces affect osteoclastogenesis and thus may be involved in the induction of local bone resorption and the formation of osteolytic lesions, leading eventually to the loosening of orthopedic implants.

Variation of cell and matrix morphologies in articular cartilage among locations in the adult human knee

OBJECTIVE: Understanding of articular cartilage physiology, remodelling mechanisms, and evaluation of tissue engineering repair methods requires reference information regarding normal structural organization. Our goals were to examine the variation of cartilage cell and matrix morphology in different topographical areas of the adult human knee joint. METHODS: Osteochondral explants were acquired from seven distinct anatomical locations of the knee joints of deceased persons aged 20-40 years and prepared for analysis of cell, matrix and tissue morphology using confocal microscopy and unbiased stereological methods. Differences between locations were identified by statistical analysis. RESULTS: Medial femoral condyle cartilage had relatively high cell surface area per unit tissue volume in the superficial zone. In the transitional zone, meniscus-covered lateral tibia cartilage showed elevated chondrocyte densities compared to the rest of the knee while lateral femoral condyle cartilage exhibited particularly large chondrocytes. Statistical analyses indicated highly uniform morphology throughout the radial zone (lower 80% of cartilage thickness) in the knee, and strong similarities in cell and matrix morphologies among cartilage from the femoral condyles and also in the mediocentral tibial plateau. Throughout the adult human knee, the mean matrix volume per chondron was remarkably constant at approximately 224,000 microm(3), corresponding to approximately 4.6 x 10(6) chondrons per cm(3). CONCLUSIONS: The uniformity of matrix volume per chondron throughout the adult human knee suggests that cell-scale biophysical and metabolic constraints may place limitations on cartilage thickness, mechanical properties, and remodelling mechanisms. Data may also aid the evaluation of cartilage tissue engineering treatments in a site-specific manner. Results indicate that joint locations which perform similar biomechanical functions have similar cell and matrix morphologies; findings may therefore also provide clues to understanding conditions under which focal lesions leading to osteoarthritis may occur.

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

BACKGROUND CONTEXT Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell-embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo-reversible hydrogel (TR-HG), based on a modified polysaccharide with a thermo-reversible polyamide [poly(N-isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain-induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR-HG; hMSCs were additionally preconditioned with rhGDF-5 for 7 days. Then, TR-HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR-HG (material control); (D4) PDDM + TR-HG + bNPCs; (D5) PDDM + TR-HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel-filled PDDM and assessment of disc height and volume changes. In gel-injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load-bearing hydrogels that are also cyto-compatible.