Fibre‐based scaffolding techniques for tendon tissue engineering

Tendon refers to a band of tough, regularly arranged, and connective tissue connecting muscle and bone, transferring strength from muscle to bone, and enabling articular stability and movement. The limitations of natural tendon grafts motivate the scaffold‐based tissue engineering (TE) approaches, which aim to build patient‐specific biological substitutes that can repair the damaged or diseased tissues. Advances in engineering and knowledge of chemistry and biology have brought forth numerous fibre‐based technologies, including electrospinning, electrohydrodynamic jet printing, electrochemical alignment technique, and other fibre‐assembly technologies, which enable the fabrication of tendon tissue structure in 3‐dimension. Textile techniques such as knitting and braiding have also been performed based on the fibrous materials to produce more complex structure. These scaffolds showed great similarity with native tendons in architectural features, mechanical properties, and facilitate biological functionality such as cellular adhesion, ingrowth, proliferation, and differentiation towards tendon tissue. Herein, we review the techniques that have been used to assemble fibres into scaffolds for tendon TE application. The morphological structures, mechanical properties, materials, degradation characteristics, and biological activities of the induced scaffolds were compared. The existing challenges and future prospects of fibre‐based tendon TE have also been discussed.     

Impact of modified gelatin on valvular microtissues

A significant challenge in the field of tissue engineering is the biofabrication of three‐dimensional (3D) functional tissues with direct applications in organ‐on‐a‐chip systems and future organ engineering. Multicellular valvular microtissues can be used as building blocks for the formation of larger scale valvular macrotissues. Yet, for the controlled biofabrication of 3D macrotissues with predefined complex shapes, directed assembly of microtissues through bioprinting is needed. This study aimed to investigate if modified gelatin is an instructive material for valvular microtissues. Valvular microtissues were encapsulated in modified gelatin hydrogels and cross‐linked in the presence of a photoinitiator (Irgacure 2959 or VA‐086). Hydrogel properties were determined, and valvular interstitial cell functions like phenotype, proliferation, migration, mRNA expression of extracellular matrix (ECM) molecules, ECM deposition, and tissue fusion were characterized by histochemical stainings and RT‐qPCR. Encapsulated microtissues remained viable, produced heart valve‐related ECM components, and remained in a quiescent state. However, encapsulation induced some changes in ECM formation and gene expression. Encapsulated microtissues showed lower remodeling capacity and increased expression levels of Col I/V, elastin, hyaluronan, biglycan, decorin, and Sox9 compared with nonencapsulated microtissues. Furthermore, this study demonstrated that proliferation, migration, and tissue fusion was more pronounced in softer gels. In general, we evidenced that modified gelatin is an instructive material for physiologically relevant valvular microtissues and provided a proof of concept for the formation of larger valvular tissue by assembling microtissues at random in soft gels.     

Cell transplantation as replacement therapy for the future.

Cell transplantation and tissue engineering techniques are being developed to generate new functional tissues for human applications. New techniques have been developed to generate new cartilage for reconstructive applications and new liver tissue as replacement therapy. Devices are constructed using synthetic polymers as scaffolding attached to tissue-specific cells, thereby allowing for tissue remodeling and functional replacement.     

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three‐dimensional prevascularized tissues for diverse applications

Self‐sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi‐potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three‐dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co‐culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O₂) was introduced during the 7 day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O₂) for up to 7 days. The results showed that a branched and mature vascular network was formed in the co‐culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin‐1 (Ang‐1) were significantly increased by low‐oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi‐potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three‐dimensional prevascularized tissues for diverse applications.     

Transplantation of three‐dimensional artificial human vascular tissues fabricated using an extracellular matrix nanofilm‐based cell‐accumulation technique

We have established a novel three‐dimensional (3D) tissue‐constructing technique, referred to as the ‘cell‐accumulation method’, which is based on the self‐assembly of cultured human cells. In this technique, cells are coated with fibronectin and gelatin to construct extracellular matrix (ECM) nanofilms and cultured to form multi‐layers in vitro. By using this method, we have successfully fabricated artificial tissues with vascular networks constructed by co‐cultivation of human umbilical vein‐derived vascular endothelial cells between multi‐layers of normal human dermal fibroblasts. In this study, to assess these engineered vascular tissues as therapeutic implants, we transplanted the 3D human tissues with microvascular networks, fabricated based on the cell‐accumulation method, onto the back skin of nude mice. After the transplantation, we found vascular networks with perfusion of blood in the transplanted graft. At the boundary between host and implanted tissue, connectivity between murine and human vessels was found. Transmission electron microscopy of the implanted artificial vascular tubules demonstrated the ultrastructural features of blood capillaries. Moreover, maturation of the vascular tissues after transplantation was shown by the presence of pericyte‐like cells and abundant collagen fibrils in the ECM surrounding the vasculature. These results demonstrated that artificial human vascular tissues constructed by our method were engrafted and matured in animal skin. In addition, the implanted artificial human vascular networks were connected with the host circulatory system by anastomosis. This method is an attractive technique for engineering prevascularized artificial tissues for transplantation. Copyright © 2015 John Wiley & Sons, Ltd.     

Abdominal wall reconstruction by a regionally distinct biocomposite of extracellular matrix digest and a biodegradable elastomer

Current extracellular matrix (ECM) derived scaffolds offer promising regenerative responses in many settings, however in some applications there may be a desire for more robust and long lasting mechanical properties. A biohybrid composite material that offers both strength and bioactivity for optimal healing towards native tissue behavior may offer a solution to this problem. A regionally distinct biocomposite scaffold composed of a biodegradable elastomer (poly(ester urethane)urea) and porcine dermal ECM gel was generated to meet this need by a concurrent polymer electrospinning/ECM gel electrospraying technique where the electrosprayed component was varied temporally during the processing. A sandwich structure was achieved with polymer fiber rich upper and lower layers for structural support and an ECM‐rich inner layer to encourage cell ingrowth. Increasing the upper and lower layer fiber content predictably increased tensile strength. In a rat full thickness abdominal wall defect model, the sandwich scaffold design maintained its thickness whereas control biohybrid scaffolds lacking the upper and lower fiber‐rich regions failed at 8 weeks. Sandwich scaffold implants also showed higher collagen content 4 and 8 weeks after implantation, exhibited an increased M2 macrophage phenotype response at later times and developed biaxial mechanical properties better approximating native tissue. By employing a processing approach that creates a sheet‐form scaffold with regionally distinct zones, it was possible to improve biological outcomes in body wall repair and provide the means for further tuning scaffold mechanical parameters when targeting other applications. Copyright © 2013 John Wiley & Sons, Ltd.     

静电纺丝在组织工程的应用：距离临床转化还有多远？

背景：静电纺丝是一种制备组织工程支架材料极有前途的技术手段。目的：综述目前静电纺丝技术在组织工程中的应用进展及其在应用中存在的主要问题。方法：应用计算机检索Medline数据库、中国知网数据库2000至2013年文章,检索关键词为“静电纺丝,组织工程;Electrospinning,tissue engineering”。结果与结论：静电纺丝技术制备的纳米纤维无纺布材料,其结构类似于细胞外基质,具有高的比表面积,可控机械性能良好,便于加工,已被广泛应用到组织工程中生物降解材料和高分子聚合物的合成领域。静电纺丝在组织工程中的应用进展快速,尤其所选用的电纺材料或者与不同技术结合的电纺。静电纺丝能将材料的性能与组织的不同形态结构相结合,一系列新的聚合物被成功引入到组织工程支架中作为细胞再生和增殖的基质,然而重要的问题是,如何控制支架与生物系统的相互作用即实现细胞的浸润性生长,如何控制孔隙大小,机械性能,毒性等,在该技术应用到真正实用的生物医学中之前仍然需要进一步研究,尤其是体内研究。     

Electrospun nanofibres to mimic natural hierarchical structure of tissues: application in musculoskeletal regeneration

Biomimetic scaffolds mimicking the natural hierarchical structure of tissues have recently attracted the interest of researchers and provide a promising strategy to resemble the nonhomogeneous property of tissues. This review provides an overview of the various hierarchical length scales in the native tissues of the musculoskeletal system. It further focuses on electrospinning as a technique to mimic the tissue structures with specific emphasis on bone. The effect of cellular alignment, infiltration, vascularisation, and differentiation in these nanostructures has also been discussed. An outline of the various additive manufacturing techniques in combination with electrospinning has been elaborated. The review concludes with the challenges and future directions to understand the intricacies of bottom‐up approach to engineer the systems at a macroscale. Copyright © 2016 John Wiley & Sons, Ltd.     

Meniscus ECM‐functionalised hydrogels containing infrapatellar fat pad‐derived stem cells for bioprinting of regionally defined meniscal tissue

Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)‐derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM‐functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad‐derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad‐derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM‐functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM‐functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox‐9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM‐functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.     

A novel culture device for the evaluation of three‐dimensional extracellular matrix materials

Cell–matrix interactions in a three‐dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell–matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface‐seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top‐surface‐seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re‐endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell–ECM interactions in 3D ECM. Copyright © 2012 John Wiley & Sons, Ltd.     

Advances in three‐dimensional bioprinting of bone: Progress and challenges

Several attempts have been made to engineer a viable three‐dimensional (3D) bone tissue equivalent using conventional tissue engineering strategies, but with limited clinical success. Using 3D bioprinting technology, scientists have developed functional prototypes of clinically relevant and mechanically robust bone with a functional bone marrow. Although the field is in its infancy, it has shown immense potential in the field of bone tissue engineering by re‐establishing the 3D dynamic micro‐environment of the native bone. Inspite of their in vitro success, maintaining the viability and differentiation potential of such cell‐laden constructs overtime, and their subsequent preclinical testing in terms of stability, mechanical loading, immune responses, and osseointegrative potential still needs to be explored. Progress is slow due to several challenges such as but not limited to the choice of ink used for cell encapsulation, optimal cell source, bioprinting method suitable for replicating the heterogeneous tissues and organs, and so on. Here, we summarize the recent advancements in bioprinting of bone, their limitations, challenges, and strategies for future improvisations. The generated knowledge will provide deep insights on our current understanding of the cellular interactions with the hydrogel matrices and help to unravel new methodologies for facilitating precisely regulated stem cell behaviour.     

Extracellular matrix administration as a potential therapeutic strategy for periodontal ligament regeneration

INTRODUCTION: The current strategies employed for the treatment of connective tissue disease include the application of stem cells, the use of functional molecules that can reorganize tissue integrity and cellular activities to recover connective tissue function. Approaches to the regeneration of periodontal tissue, which is the tooth-supporting connective tissue, have made some progress recently and provide a useful experimental model for the evaluation of future strategies to treat connective tissue diseases such as periodontal disease. AREAS COVERED: The ultimate goal of periodontal tissue regeneration is to reconstruct the ligament structure that will sustain the required mechanical force to connect with mineralized tissues such as cementum and alveolar bone. In this review, we discuss the proposed use of extracellular matrix (ECM) administration therapy as an additional therapeutic strategy to stem cell transplantation and cytokine administration in the current field of periodontal tissue regeneration therapy. EXPERT OPINION: Although various available tissue engineering technologies can now achieve periodontal tissue regeneration, ECM administration therapy is likely to play an essential future role in the development and regeneration of periodontal tissue and attenuate the signaling events that mediate tissue degradation. Hence, ECM administration could serve as a novel technology in periodontal tissue regeneration and also as a viable approach to alleviating connective tissue disorders such as Marfan's syndrome.     

Development of decellularized scaffolds for stem cell‐driven tissue engineering

Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplantation leads to the death of numerous patients waiting for a suitable organ donor. The potential of decellularized scaffolds, derived from native tissues or organs in the form of scaffolds has been evolved as a promising approach in tissue‐regenerative medicine for translating functional organ replacements. In recent years, donor organs, such as heart, liver, lung and kidneys, have been reported to provide acellular extracellular matrix (ECM)‐based scaffolds through the process called ‘decellularization’ and proved to show the potential of recellularization with selected cell populations, particularly with stem cells. In fact, decellularized stem cell matrix (DSCM) has also emerged as a potent biological scaffold for controlling stem cell fate and function during tissue organization. Despite the proven potential of decellularized scaffolds in tissue engineering, the molecular mechanism responsible for stem cell interactions with decellularized scaffolds is still unclear. Stem cells interact with, and respond to, various signals/cues emanating from their ECM. The ability to harness the regenerative potential of stem cells via decellularized ECM‐based scaffolds has promising implications for tissue‐regenerative medicine. Keeping these points in view, this article reviews the current status of decellularized scaffolds for stem cells, with particular focus on: (a) concept and various methods of decellularization; (b) interaction of stem cells with decellularized scaffolds; (c) current recellularization strategies, with associated challenges; and (iv) applications of the decellularized scaffolds in stem cell‐driven tissue engineering and regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

Multiphoton microscopy analysis of extracellular collagen I network formation by mesenchymal stem cells

Collagen I is the major fibrous extracellular component of bone responsible for its ultimate tensile strength. In tissue engineering, one of the most important challenges for tissue formation is to get cells interconnected via a strong and functional extracellular matrix (ECM), mimicking as closely as possible the natural ECM geometry. Still missing in tissue engineering are: (a) a versatile, high‐resolution and non‐invasive approach to evaluate and quantify different aspects of ECM development within engineered biomimetic scaffolds online; and (b) deeper insights into the mechanism whereby cellular matrix production is enhanced in 3D cell–scaffold composites, putatively via enhanced focal adhesion linkage, over the 2D setting. In this study, we developed sensitive morphometric detection methods for collagen I‐producing and bone‐forming mesenchymal stem cells (MSCs), based on multiphoton second harmonic generation (SHG) microscopy, and used those techniques to compare collagen I production capabilities in 2D‐ and 3D‐arranged cells. We found that stimulating cells with 1% serum in the presence of ascorbic acid is superior to other medium conditions tested, including classical osteogenic medium. In contrast to conventional 2D culture, having MSCs packed closely in a 3D environment presumably stimulates cells to produce strong and complex collagen I networks with defined network structures (visible in SHG images) and improves collagen production. Copyright © 2015 John Wiley & Sons, Ltd.     

Vascular tissue engineering of small‐diameter blood vessels: reviewing the electrospinning approach

Vascular tissue engineering is a relevant research field aimed at elaborating and proposing innovative solutions to overcome the drawbacks related to the use of conventional blood vessel substitutes, especially referring to small‐diameter grafts. For this aim, electrospinning can be regarded as a valuable technique to produce novel scaffolds with several functional characteristics that can be usefully tailored for the application discussed here. The reproduction of the natural extracellular matrix obtained by processing bioresorbable polymers, either functionalized or not, is driving the biomedical research towards technical solutions that can lead to an actual therapeutic improvement. In this context, this paper reviews those studies focused on the selection of suitable biomaterials for vascular applications, their microstructure, the cell response to polymeric fibres and the strategies considered so far to modify and therefore enhance the performance of final electrospun scaffolds. Copyright © 2013 John Wiley & Sons, Ltd.     

Hydroxyapatite‐intertwined hybrid nanofibres for the mineralization of osteoblasts

Advances in tissue engineering have enabled the development of bioactive composite materials to generate biomimetic nanofibrous scaffolds for bone replacement therapies. Polymeric biocomposite nanofibrous scaffolds architecturally mimic the native extracellular matrix (ECM), delivering tremendous regenerative potential for bone tissue engineering. In the present study, biocompatible poly(l ‐lactic acid)‐co‐poly(ε‐caprolactone)–silk fibroin–hydroxyapatite–hyaluronic acid (PLACL–SF–HaP–HA) nanofibrous scaffolds were fabricated by electrospinning to mimic the native ECM. The developed nanofibrous scaffolds were characterized in terms of fibre morphology, functional group, hydrophilicity and mechanical strength, using SEM, FTIR, contact angle and tabletop tensile‐tester, respectively. The nanofibrous scaffolds showed a higher level of pore size and increased porosity of up to 95% for the exchange of nutrients and metabolic wastes. The fibre diameters obtained were in the range of around 255 ± 13.4–789 ± 22.41 nm. Osteoblasts cultured on PLACL–SF–HaP–HA showed a significantly (p < 0.001) higher level of proliferation (53%) and increased osteogenic differentiation and mineralization (63%) for the inclusion of bioactive molecules SF–HA. Energy‐dispersive X‐ray analysis (EDX) data proved that the presence of calcium and phosphorous in PLACL–SF–HaP–HA nanofibrous scaffolds was greater than in the other nanofibrous scaffolds with cultured osteoblasts. The obtained results for functionalized PLACL–SF–HaP–HA nanofibrous scaffolds proved them to be a potential biocomposite for bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Fabrication of blood‐derived elastogenic vascular grafts using electrospun fibrinogen and polycaprolactone composite scaffolds for paediatric applications

The development of tissue‐engineered vascular grafts (TEVGs) for paediatric applications must consider unique factors associated with this patient cohort. Although the increased elastogenic potential of neonatal cells offers an opportunity to overcome the long‐standing challenge of in vitro elastogenesis, neonatal patients have a lower tolerance for autologous tissue harvest and require grafts that exhibit growth potential. The purpose of this study was to apply a multipronged strategy to promote elastogenesis in conjunction with umbilical cord‐derived materials in the production of a functional paediatric TEVG. An initial proof‐of‐concept study was performed to extract fibrinogen from human umbilical cord blood samples and, through electrospinning, to produce a nanofibrous fibrinogen scaffold. This scaffold was seeded with human umbilical artery‐derived smooth muscle cells (hUASMCs), and neotissue formation within the scaffold was examined using immunofluorescence microscopy. Subsequently, a polycaprolactone‐reinforced porcine blood‐derived fibrinogen scaffold (isolated using the same protocol as cord blood fibrinogen) was used to develop a rolled‐sheet graft that employed topographical and biochemical guidance cues to promote elastogenesis and cellular orientation. This approach resulted in a TEVG with robust mechanical properties and biomimetic arrangement of extracellular matrix (ECM) with rich expression of elastic fibre‐related proteins. The results of this study hold promise for further development of paediatric TEVGs and the exploration of the effects of scaffold microstructure and nanostructure on vascular cell function and ECM production.     

De novo production of human extracellular matrix supports increased throughput and cellular complexity in 3D skin equivalent model

Three‐dimensional (3D) tissue models of human skin are being developed to better understand disease phenotypes and to screen new drugs for potential therapies. Several factors will increase the value of these in vitro 3D skin tissues for these purposes. These include the need for human‐derived extracellular matrix (ECM), higher throughput tissue formats, and greater cellular complexity. Here, we present an approach for the fabrication of 3D skin‐like tissues as a platform that addresses these three considerations. We demonstrate that human adult and neonatal fibroblasts deposit an endogenous ECM de novo that serves as an effective stroma for full epithelial tissue development and differentiation. We have miniaturized these tissues to a 24‐well format to adapt them for eventual higher throughput drug screening. We have shown that monocytes from the peripheral blood can be incorporated into this model as macrophages to increase tissue complexity. This humanized skin‐like tissue decreases dependency on animal‐derived ECM while increasing cellular complexity that can enable screening inflammatory responses in tissue models of human skin.     

组织工程肺的细胞源及支撑架

背景：虽然组织工程肺组织研究进展缓慢,但是应用干细胞或祖细胞及相关支撑架在一定条件下培育出功能健全的可用来替换的肺组织,有望为许多肺部疾患的肺移植治疗提供供体。目的：综述组织工程肺研究中肺的细胞源及支撑架的研究进展。方法：应用计算机检索中国期刊全文数据库和PubMed数据库（1990-01/2009-01）相关文献,检索词分别为：＂组织工程肺;细胞源;支撑架＂和＂tissue-engineered lung;cell source;support scaffolding＂,语言分别设定为中文和英文,共检索到89篇文章,阅读文题和摘要进行筛选,选择具有原创性,论点论据可靠且分析全面的组织工程肺细胞源及支撑架密切相关文章,排除重复性研究以及质量较差文章,最后纳入33篇进行总结综述。结果与结论：由于肺组织结构复杂、再生能力有限,肺疾病的治疗已成为棘手的问题。目前认为组织工程可培育出功能健全的可用来替换的肺组织。这是一种很有前途的治疗手段,但目前尚处于研究的初级阶段。相信随着组织工程肺的细胞源及支撑架研究的深入,会有更多的新发现,组织工程肺研究在临床应用上具有广阔的前景。     

Bioengineered post‐natal recombinant tooth bud models

The long‐term goal of this study is to devise reliable methods to regenerate full‐sized and fully functional biological teeth in humans. In this study, three‐dimensional (3D) tissue engineering methods were used to characterize intact postnatal dental tissue recombinant constructs, and dental cell suspension recombinant constructs, as models for bioengineered tooth development. In contrast to studies using mouse embryonic dental tissues and cells, here the odontogenic potential of intact dental tissues and dental cell suspensions harvested from post natal porcine teeth and human third molar wisdom tooth dental pulp were examined. The recombinant 3D tooth constructs were cultured in osteogenic media in vitro for 1 week before subcutaneous transplantation in athymic nude rat hosts for 1 month or 3 months. Subsequent analyses using X‐ray, histological and immunohistochemical methods showed that the majority of the recombinant tooth structures formed calcified tissues, including osteodentin, dentin cementum, enamel and morphologically typical tooth crowns composed of dentin and enamel. The demonstrated formation of mineralized dental tissues and tooth crown structures from easily obtained post‐natal dental tissues is an important step toward reaching the long‐term goal of establishing robust and reliable models for human tooth regeneration. Copyright © 2014 John Wiley & Sons, Ltd.