Differentiation of multipotent mesenchymal stromal cells of bone marrow into cells of cartilage tissue by culturing in three-demential OPLA scaffolds

Bone marrow multipotent mesenchymal stromal cells represent a perspective material for engineering of human three-dimensional transplants of cartilage tissue. We are demonstrated the opportunity of the directed differentiation of BM MMSC in cells of cartilage tissue by culturing them in three-dimensional scaffolds, presented by polymer OPLA in medium with inductors of chondrogenesis. For loading cells in porous scaffolds used method which essence consist in saturation of polymeric blocks by cellular suspension with the subsequent centrifugal force of cells in scaffolds and culturing of engineering constructs for 28 days in chondrogenic medium. Histological analysis derived in vitro of three-dimensional transplants showed uniform distribution of cells in the matrix with morphologically distinct chondrocytes-like cells of hyaline cartilage. Immunohistochemical analysis detected aggrecan and collagen type II within the extracellular matrix. Preclinical the researches lead on a livestock of immunodeficient mice have shown not toxicity of the engineering constructs.     

Chondrogenesis and developments in our understanding

Conditions affecting cartilage through damage or age-related degeneration pose significant challenges to individual patients and their healthcare systems. The disease burden will rise in the future as life expectancy increases. This has resulted in vigorous efforts to develop novel therapies to meet current and future needs. Due to the limited regenerative capacity of cartilage, in vitro tissue engineering techniques have emerged as the favoured technique by which to develop replacements. Tissue engineering is mainly concerned with developing cartilage replacements in the form of chondrocyte suspensions and three-dimensional scaffolds seeded with chondrocytes. One major limiting factor in the development of clinically useful cartilage constructs is our understanding of the process by which cartilage is formed, chondrogenesis. For example, techniques of culturing chondrocytes in vitro have been used for decades, resulting in chondrocyte-like cells which produce an extracellular matrix of similar composition to native cartilage, but with inferior physical properties. It has now been realised that one aspect of chondrogenesis which had been ignored was the physical context in which cartilage exists in vivo. This has resulted in the development of bioreactor systems which aim to introduce various physical stresses to engineered cartilage in a controlled environment. This has resulted in some improvements in the quality of tissue engineered cartilage. This is but one example of how the knowledge of chondrogenesis has been translated into research practice. This paper aims to review what is currently known about the process of chondrogenesis and discusses how this knowledge can be applied to tissue engineering.     

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.     

The Effect of 3D Nanofibrous Scaffolds on the Chondrogenesis of Induced Pluripotent Stem Cells and Their Application in Restoration of Cartilage Defects

The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.     

Microgravity tissue engineering

Summary Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter × 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.     

The restoration of full-thickness cartilage defects with mesenchymal stem cells (MSCs) loaded and cross-linked bilayer collagen scaffolds on rabbit model

Cartilage has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic. This study aimed to investigate the effect of mesenchymal stem cells (MSCs) loaded three-dimensional bilayer collagen scaffold for cartilage repair. Cross-linked three-dimensional bilayer collagen scaffolds seeded with or without MSCs were implanted into large cartilage defects (4 mm in diameter; 3 mm in depth) in rabbit knees. The untreated cartilage defects served as control. The tissue response was evaluated at 6 and 12 weeks after implantation by general histology and semi-quantitative histological grading systems. In addition, the repaired tissues were evaluated by mechanical test at 12 weeks after implantation. The MSCs-loaded collagen scaffold group showed the most hyaline cartilage, highest histological scores and compressive modulus. Moreover, it showed a good integration with the subchondral bone and adjacent cartilage. The structure of the novel bilayer collagen scaffolds provided architectural support for the differentiation of MSCs and demonstrated successful induction of in vivo chondrogenesis. These findings suggested that MSCs-loaded bilayer collagen scaffold could be an appealing candidate to be used for cartilage regeneration.     

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.     

Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering

A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.     

Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation

One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three-dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long-term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non-woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30-day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: (1) free-swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Biophysical regulation during cardiac development and application to tissue engineering

Tissue engineering combines the principles of biology, engineering and medicine to create biological substitutes of native tissues, with an overall objective to restore normal tissue function. It is thought that the factors regulating tissue development in vivo (genetic, molecular and physical) can also direct cell fate and tissue assembly in vitro. In light of this paradigm, tissue engineering can be viewed as an effort of "imitating nature". We first discuss biophysical regulation during cardiac development and the factors of interest for application in tissue engineering of the myocardium. Then we focus on the biomimetic approach to cardiac tissue engineering which involves the use of culture systems designed to recapitulate some aspects of the actual in vivo environment. To mimic cell signaling in native myocardium, subpopulations of neonatal rat heart cells were cultured at a physiologically high cell density in three-dimensional polymer scaffolds. To mimic the capillary network, highly porous elastomer scaffolds with arrays of parallel channels were perfused with culture medium. To mimic oxygen supply by hemoglobin, culture medium was supplemented with an oxygen carrier. To enhance electromechanical coupling, tissue constructs were induced to contract by applying electrical signals mimicking those in native heart. Over only eight days of cultivation, the biomimetic approach resulted in tissue constructs which contained electromechanically coupled cells expressing cardiac differentiation markers and cardiac-like ultrastructure and contracting synchronously in response to electrical stimulation. Ongoing studies are aimed at extending this approach to tissue engineering of functional cardiac grafts based on human cells.     

Dynamic compression of rabbit adipose-derived stem cells transfected with insulin-like growth factor 1 in chitosan/gelatin scaffolds induces chondrogenesis and matrix biosynthesis

Articular cartilage is routinely subjected to mechanical forces and growth factors. Adipose-derived stem cells (ASCs) are multi-potent adult stem cells and capable of chondrogenesis. In the present study, we investigated the comparative and interactive effects of dynamic compression and insulin-like growth factor-I (IGF-I) on the chondrogenesis of rabbit ASCs in chitosan/gelatin scaffolds. Rabbit ASCs with or without a plasmid overexpressing of human IGF-1 were cultured in chitosan/gelatin scaffolds for 2 days, then subjected to cyclic compression with 5% strain and 1 Hz for 4 h per day for seven consecutive days. Dynamic compression induced chondrogenesis of rabbit ASCs by activating calcium signaling pathways and up-regulating the expression of Sox-9. Dynamic compression plus IGF-1 overexpression up-regulated expression of chondrocyte-specific extracellular matrix genes including type II collagen, Sox-9, and aggrecan with no effect on type X collagen expression. Furthermore, dynamic compression and IGF-1 expression promoted cellular proliferation and the deposition of proteoglycan and collagen. Intracellular calcium ion concentration and peak currents of Ca(2+) ion channels were consistent with chondrocytes. The tissue-engineered cartilage from this process had excellent mechanical properties. When applied together, the effects achieved by the two stimuli (dynamic compression and IGF-1) were greater than those achieved by either stimulus alone. Our results suggest that dynamic compression combined with IGF-1 overexpression might benefit articular cartilage tissue engineering in cartilage regeneration.     

Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.     

Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling

The supply of oxygen within three-dimensional tissue-engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2-5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 x 10(-9)) + (0.4 x 10(-9) m(2) s(-1)), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 x 10(-10) m(2) s(-1)). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 x 10(-19) mol m(-3) s(-1)), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis.     

Osteogenic differentiation and osteochondral tissue engineering using human adipose‐derived stem cells

Osteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose‐derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual‐chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1‐fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual‐chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem‐cell source. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating, in vitro, tissues with more authentic properties. Cell clusters called spheroids are the basis for scaffold-free tissue engineering. In this review, we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues. Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis, and are capable of spontaneously fusing to other spheroids, making them ideal building blocks for bone and cartilage tissue engineering. Here, we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro. Overall, recent studies support the notion that spheroids are ideal "building blocks" for tissue engineering by “bottom-up” approaches, which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting. Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.     

Tissue transglutaminase_variant 2-transduced mesenchymal stem cells and their chondrogenic potential

As cartilage is incapable of self-healing upon severe degeneration because of the lack of blood vessels, cartilage tissue engineering is gaining importance in the treatment of cartilage defects. This study was designed to improve cartilage tissue regeneration by expressing tissue transglutaminase variant 2 (TGM2_v2) in mesenchymal stem cells (MSC) derived from bone marrow of rats. For this purpose, rat MSCs transduced with TGM2_v2 were grown and differentiated on three-dimensional polybutylene succinate (PBSu) and poly-l -lactide (PLLA) blend scaffolds. The transduced cells could not only successfully express the short form transglutaminase-2, but also deposited the protein onto the scaffolds. In addition, they could spontaneously produce cartilage-specific proteins without any chondrogenic induction, suggesting that TGM2_v2 expression provided the cells the ability of chondrogenic differentiation. PBSu:PLLA scaffolds loaded with TGM2_v2 expressing MSCs could be used in repair of articular cartilage defects.     

Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen ( approximately 5%) environment in vivo; therefore, oxygen tension may be an important factor that regulates the metabolism of chondrocyte progenitors. A number of recent studies have developed tissue engineering approaches for promoting cartilage repair using undifferentiated progenitor cells seeded on biomaterial scaffolds, but little is known about how oxygen might influence these engineered tissues. Human adipose-derived adult stem (hADAS) cells isolated from the stroma of subcutaneous fat were suspended in alginate beads and cultured in control or chondrogenic media in either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days. Under chondrogenic conditions, low oxygen tension significantly inhibited the proliferation of hADAS cells, but induced a two-fold increase in the rate of protein synthesis and a three-fold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain timepoints. Immunohistochemical analysis showed significant production of cartilage-associated matrix molecules, including collagen type II and chondroitin-4-sulfate. These findings suggest oxygen tension may play an important role in regulating the proliferation and metabolism of hADAS cells as they undergo chondrogenesis, and the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue-engineered cartilage.     

Engineering cartilage tissues with the shape of human nasal alar by using chondrocyte macroaggregate--Experiment study in rabbit model

Despite of progresses in tissue engineering based on cell/scaffold strategy, uneven cell distribution as well as tissue formation in the scaffold, limited cell seeding efficiency and inflammatory reaction triggered by the degradation of scaffold remain problems to be resolved. In this study, we proposed a novel cell-macroaggregate cultivation system, and explored a feasible strategy to construct three-dimensional cartilage tissue with shape of human nasal alar by using cell macroaggregate. Isolated chondrocytes was cultured at high density to form a monolayer chondrocyte sheet as well as expanded for seeding on the sheet to produce mechanically operable cell macroaggregate. Chondrocyte macroaggregates were then fabricated into transplants with shape of nasal alar by using Internal support or External scaffold techniques; results of in vivo chondrogenesis were investigated in immunocompetent animal. Chondrocyte macroaggregates presented long survival time and good viability; constructs fabricated using both techniques can develop into tissues with characteristic structure of native cartilage, glycosaminoglycans as well as type II collagen were highly produced in the ECM of engineered cartilages. By placing hyaluronan ester film as Internal support, the predetermined shape of the chondrocyte macroaggregate can be well maintained. In contrast, due to the poor mechanical stability of grafts fabricated in External scaffold group, obvious deformation occurred in harvested specimens. The experiment proved the usefulness of chondrocyte macroaggregate in cartilage regeneration, and provided a new strategy to engineer cartilage with special shape by using cell macroaggregate/biodegradable support.