Design and validation of a dynamic cell‐culture system for bone biology research and exogenous tissue‐engineering applications

Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell‐culture methods are ill‐suited to study the intricacies of bone biology because they ignore the three‐dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2–3 mm thickness. In this project, a flow‐perfusion system was custom‐designed to overcome these limitations: eight adaptable chambers housed cylindrical cell‐seeded scaffolds measuring 12 or 24 mm in diameter and 1–10 mm in thickness. The porous scaffolds were manufactured using a three‐dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi‐channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas‐permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor–scaffold system, using 12 × 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright © 2013 John Wiley & Sons, Ltd.     

Nanopatterned collagen tubes for vascular tissue engineering

Nanopatterned (330 nm wide channels) type I collagen films were prepared by solvent casting on poly(dimethyl siloxane) (PDMS) templates. These films were rolled into tubular constructs and crosslinked. Tubular constructs were incubated under cell culture conditions for 28 days and examined by stereomicroscopy and scanning electron microscopy (SEM) for the integrity of the structure. The nanopatterned films were also seeded with human vascular smooth muscle cells (VSMCs) and examined after immunostaining with fluorescence microscopy and SEM to assess the cell phenotype and alignment on the nanopatterns on the films.     

Aligned electrospun scaffolds and elastogenic factors for vascular cell‐mediated elastic matrix assembly

Strategies to enhance the production of organized elastic matrix by smooth muscle cells (SMCs) are critical in engineering functional vascular conduits. Therefore, the goal of this study was to determine the effect of different surfaces, i.e. random and aligned electrospun poly(ε‐caprolactone) meshes and two‐dimensional (2D) controls, and exogenous elastogenic factors on the cultured rat aortic SMC phenotype and production of extracellular matrix. This study demonstrated that aligned electrospun fibres guide cell alignment, induce a more elongated cell morphology and promote a more synthetic phenotype. Importantly, these cells produced greater amounts of elastin‐rich matrix per cell on the electrospun scaffolds. In addition, exogenous elastogenic factors severely limited rat aortic smooth muscle cells (RASMCs) proliferation and promoted a more synthetic SMC phenotype on electrospun meshes, but they had less effect on 2D controls. Finally, the elastogenic factors induced the SMCs to generate more matrix collagen and elastin on a per cell basis. Together, these results demonstrate the elastogenic benefits of electrospun meshes. Copyright © 2011 John Wiley & Sons, Ltd.     

Spray‐ and laser‐assisted biomaterial processing for fast and efficient autologous cell‐plus‐matrix tissue engineering

At present, intensive investigation aims at the creation of optimal valvular prostheses. We introduced and tested the applicability and functionality of two advanced cell‐plus‐matrix seeding technologies, spray‐assisted bioprocessing (SaBP) and laser‐assisted bioprocessing (LaBP), for autologous tissue engineering (TE) of bioresorbable artificial grafts. For SaBP, human mesenchymal stem cells (HMSCs), umbilical cord vein endothelial cells (HUVECs) and fibrin were simultaneously spray‐administered on poly(ε‐caprolactone) (PCL) substrates. For LaBP, HUVECs and HMSCs were separately laser‐printed in stripes, followed by fibrin sealing. Three‐leaflet valves were manufactured following TE of electrospun PCL tissue equivalents. Grafts were monitored in vitro under static and dynamic conditions in bioreactors. SaBP and LaBP resulted in TE of grafts with homogeneous cell distribution and accurate cell pattern, respectively. The engineered valves demonstrated immediate sufficient performance, complete cell coating, proliferation, engraftment, HUVEC‐mediated invasion, HMSC differentiation and extracellular matrix deposition. SaBP revealed higher efficiency, with at least 12‐fold shorter processing time than the applied LaBP set‐up. LaBP realized coating with higher cell density and minimal cell–scaffold distance. Fibrin and PCL stability remain issues for improvement. The introduced TE technologies resulted in complete valvular cell‐plus‐matrix coating, excellent engraftment and HMSCs differentiation. SaBP might have potential for intraoperative table‐side TE considering the procedural duration and ease of implementation. LaBP might accelerate engraftment with precise patterns. Copyright © 2012 John Wiley & Sons, Ltd.     

Omental grafting: a cell‐based therapy for blood vessel repair

Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin β4 (Tβ4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tβ4‐induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tβ4‐stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tβ4 promoted proliferation, migration and trans‐differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell‐based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm‐setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields. Copyright © 2012 John Wiley & Sons, Ltd.     

Smooth muscle tissue engineering for hybrid tubular organs: scanning electron microscopic investigations of cell interactions with collagen scaffolds

Tissue engineering of transplantable tubular tissue necessitates a composite approach to assembling the individual components. In the engineering of tubular structures (gastrointestinal, urogenital and vascular tissues) there is a demand for smooth muscle tissue in addition to the primary organ‐specific tissue. This study aims to investigate the interaction of smooth muscle cells on bovine collagen scaffolds cross‐linked with glutaraldehyde under in vitro conditions, using scanning electron microscopy. The density of tissue generated over the 8 week period was demonstrated in terms of: (a) scaffold coverage and attachment; (b) cell density; (c) cell viability deep inside the scaffolds; and (d) cell differentiation. Copyright © 2009 John Wiley & Sons, Ltd.     

Towards embryonic‐like scaffolds for skin tissue engineering: identification of effector molecules and construction of scaffolds

Autologous skin grafts are the gold standard for the treatment of burn wounds. In a number of cases, treatment with autologous tissue is not possible and skin substitutes are used. The outcome, however, is not optimal and improvements are needed. Inspired by scarless healing in early embryonic development, we here set out a strategy for the design and construction of embryonic‐like scaffolds for skin tissue engineering. This strategy may serve as a general approach in the construction of embryonic‐like scaffolds for other tissues/organ. As a first step, key effector molecules upregulated during embryonic and neonatal skin formation were identified using a comparative gene expressing analysis. A set of 20 effector molecules was identified, from which insulin‐like growth factor 2 (IGF2) and sonic hedgehog (SHH) were selected for incorporation into a type I collagen–heparin scaffold. Porous scaffolds were constructed using purified collagen fibrils and 6% covalently bound heparin (to bind and protect the growth factors), and IGF2 and SHH were incorporated either individually (~0.7 and 0.4 µg/mg scaffolds) or in combination (combined ~1.5 µg/mg scaffolds). In addition, scaffolds containing hyaluronan (up to 20 µg/mg scaffold) were prepared, based on the up‐ or downregulation of genes involved in hyaluronan synthesis/degradation and its suggested role in scarless healing. In conclusion, based on a comprehensive gene expression analysis, a set of effector molecules and matrix molecules was identified and incorporated into porous scaffolds. The scaffolds thus prepared may create an 'embryonic‐like' environment for cells to recapitulate embryonic events and for new tissues/organs. Copyright © 2013 John Wiley & Sons, Ltd.     

Efficient in vivo vascularization of tissue‐engineering scaffolds

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra‐scaffold microvessel self‐assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly‐L ‐lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra‐scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue‐engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel‐enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra‐scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non‐random and displayed a preferential perivascular localization. Hence, efficient microvessel self‐assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of chitosan–gelatin scaffolds for dermal tissue engineering

Porous scaffolds for dermal tissue engineering were fabricated by freeze‐drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1‐(3‐dimethylaminopropyl)‐3‐ethyl‐carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well‐distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin‐based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue‐engineered skin. Copyright © 2011 John Wiley & Sons, Ltd.     

A collagen network phase improves cell seeding of open‐pore structure scaffolds under perfusion

Scaffolds with open‐pore morphologies offer several advantages in cell‐based tissue engineering, but their use is limited by a low cell‐seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open‐pore architecture of polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds increases human bone marrow stromal cells (hBMSCs) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL–TCP scaffolds, rapid prototyped with a honeycomb‐like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen‐free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSCs directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSCs under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5‐fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non‐crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell‐based therapies, collagen‐filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion‐based bioreactor devices to streamline graft manufacture. Copyright © 2011 John Wiley & Sons, Ltd.     

Porous polylactide/β‐tricalcium phosphate composite scaffolds for tissue engineering applications

Porous polylactide/β‐tricalcium phosphate (PLA/β‐TCP) composite scaffolds were fabricated by freeze‐drying. The aim of this study was to characterize these graded porous composite scaffolds in two different PLA concentrations (2 and 3 wt%). Also, three different β‐TCP ratios (5, 10 and 20 wt%) were used to study the effect of β‐TCP on the properties of the polymer. The characterization was carried out by determining the pH, weight change, component ratios, thermal stability, inherent viscosity and microstructure of the scaffolds in 26 weeks of hydrolysis. This study indicated that no considerable change was noticed in the structure of the scaffolds when the β‐TCP filler was added. Also, the amount of β‐TCP did not affect the pore size or the pore distribution in the scaffolds. We observed that the fabrication method improved the thermal stability of the samples. Our results suggest that, from the structural point of view, these scaffolds could have potential for the treatment of osteochondral defects in tissue engineering applications. The porous bottom surface of the scaffold and the increased osteogenic differentiation potential achieved with β‐TCP particles may encourage the growth of bone cells. In addition, the dense surface skin of the scaffold may inhibit the ingrowth of osteoblasts and bone tissue, while simultaneously encouraging the ingrowth of chondrocytes. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of novel akermanite:poly‐ϵ‐caprolactone scaffolds for human adipose‐derived stem cells bone tissue engineering

In this study, three different akermanite:poly‐?‐caprolactone (PCL) composite scaffolds (wt%: 75:25, 50:50, 25:75) were characterized in terms of structure, compression strength, degradation rate and in vitro biocompatibility to human adipose‐derived stem cells (hASC). Pure ceramic scaffolds [CellCeram^TM, custom‐made, 40:60 wt%; β‐tricalcium phosphate (β‐TCP):hydroxyapatite (HA); and akermanite] and PCL scaffolds served as experimental controls. Compared to ceramic scaffolds, the authors hypothesized that optimal akermanite:PCL composites would have improved compression strength and comparable biocompatibility to hASC. Electron microscopy analysis revealed that PCL‐containing scaffolds had the highest porosity but CellCeram^TM had the greatest pore size. In general, compression strength in PCL‐containing scaffolds was greater than in ceramic scaffolds. PCL‐containing scaffolds were also more stable in culture than ceramic scaffolds. Nonetheless, mass losses after 21 days were observed in all scaffold types. Reduced hASC metabolic activity and increased cell detachment were observed after acute exposure to akermanite:PCL extracts (wt%: 75:25, 50:50). Among the PCL‐containing scaffolds, hASC cultured for 21 days on akermanite:PCL (wt%: 75:25) discs displayed the highest viability, increased expression of osteogenic markers (alkaline phosphatase and osteocalcin) and lowest IL‐6 expression. Together, the results indicate that akermanite:PCL composites may have appropriate mechanical and biocompatibility properties for use as bone tissue scaffolds. Copyright © 2012 John Wiley & Sons, Ltd.     

Fibrous biodegradable l‐alanine‐based scaffolds for vascular tissue engineering

In vascular tissue engineering, three‐dimensional (3D) biodegradable scaffolds play an important role in guiding seeded cells to produce matrix components by providing both mechanical and biological cues. The objective of this work was to fabricate fibrous biodegradable scaffolds from novel poly(ester amide)s (PEAs) derived from l ‐alanine by electrospinning, and to study the degradation profiles and its suitability for vascular tissue‐engineering applications. In view of this, l ‐alanine‐derived PEAs (dissolved in chloroform) were electrospun together with 18–30% w/w polycaprolactone (PCL) to improve spinnability. A minimum of 18% was required to effectively electrospin the solution while the upper value was set in order to limit the influence of PCL on the electrospun PEA fibres. Electrospun fibre mats with average fibre diameters of ~0.4 µm were obtained. Both fibre diameter and porosity increased with increasing PEA content and solution concentration. The degradation of a PEA fibre mat over a period of 28 days indicated that mass loss kinetics was linear, and no change in molecular weight was found, suggesting a surface erosion mechanism. Human coronary artery smooth muscle cells (HCASMCs) cultured for 7 days on the fibre mats showed significantly higher viability (p < 0.0001), suggesting that PEA scaffolds provided a better microenvironment for seeded cells compared with control PCL fibre mats of similar fibre diameter and porosity. Furthermore, elastin expression on the PEA fibre mats was significantly higher than the pure PEA discs and pure PCL fibre mat controls (p < 0.0001). These novel biodegradable PEA fibrous scaffolds could be strong candidates for vascular tissue‐engineering applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Motility imaging via optical coherence phase microscopy enables label‐free monitoring of tissue growth and viability in 3D tissue‐engineering scaffolds

As the field of tissue engineering continues to progress, there is a deep need for non‐invasive, label‐free imaging technologies that can monitor tissue growth and health within thick three‐dimensional (3D) constructs. Amongst the many imaging modalities under investigation, optical coherence tomography (OCT) has emerged as a promising tool, enabling non‐destructive in situ characterization of scaffolds and engineered tissues. However, the lack of optical contrast between cells and scaffold materials using this technique remains a challenge. In this communication, we show that mapping the optical phase fluctuations resulting from cellular viability and motility allows for the distinction of live cells from their surrounding scaffold environment. Motility imaging was performed via a common‐path optical coherence phase microscope (OCPM), an OCT modality that has been shown to be sensitive to nanometer‐level fluctuations. More specifically, we examined the development of human adipose‐derived stem cells and/or murine pre‐osteoblasts within two distinct scaffold systems, commercially available alginate sponges and custom‐microfabricated poly(d , l ‐lactic‐co‐glycolic acid) fibrous scaffolds. Cellular motility is demonstrated as an endogenous source of contrast for OCPM, enabling real‐time, label‐free monitoring of 3D engineered tissue development. Copyright © 2013 John Wiley & Sons, Ltd.     

Cell‐laden biphasic scaffolds with anisotropic structure for the regeneration of osteochondral tissue

Sufficient treatment of chondral and osteochondral defects to restore function of the respective tissue remains challenging in regenerative medicine. Biphasic scaffolds that mimic properties of bone and cartilage are appropriate to regenerate both tissues at the same time. The present study describes the development of biphasic, but monolithic scaffolds based on alginate, which are suitable for embedding of living cells in the chondral part. Scaffolds are fabricated under sterile and cell‐compatible conditions according to the principle of diffusion‐controlled, directed ionotropic gelation, which leads to the formation of channel‐like, parallel aligned pores, running through the whole length of the biphasic constructs. The synthesis process leads to an anisotropic structure, as it is found in many natural tissues. The two different layers of the scaffolds are characterized by different microstructure and mechanical properties which provide a suitable environment for cells to form the respective tissue. Human chondrocytes and human mesenchymal stem cells were embedded within the chondral layer of the biphasic scaffolds during hydrogel formation and their chondrogenic (re)differentiation was successfully induced. Whereas viability of non‐induced human mesenchymal stem cells decreased during culture, cell viability of human chondrocytes and chondrogenically induced human mesenchymal stem cells remained high within the scaffolds over the whole culture period of 3 weeks, demonstrating successful fabrication of cell‐laden centimetre‐scaled constructs for potential application in regenerative treatment of osteochondral defects. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of the potential of novel PCL-PPDX biodegradable scaffolds as support materials for cartilage tissue engineering

Cartilage is a specialized tissue represented by a group of particular cells (the chondrocytes) and an abundant extracellular matrix. Because of the reduced regenerative capacity of this tissue, cartilage injuries are often difficult to handle. Nowadays tissue engineering has emerged as a very promising discipline, and biodegradable polymeric scaffolds are widely used as tissue supports. In cartilage injuries, the use of autologous chondrocyte implantation from non-affected cartilage zones has emerged as a very interesting technique, where chondrocytes are expanded in order to obtain a greater number of cells. Nevertheless, it has been reported that chondrocytes in bidimensional cultures suffer a dedifferentiation process. The present study sought, in the first place, to standardize a novel protocol in order to obtain primary cultures of chondrocytes from newborn rabbit hyaline cartilage from the xiphoid process. Second, the potential of porous three-dimensional (3D) biodegradable polymeric matrices as support materials for chondrocytes was evaluated: a novel poly(ε-caprolactone)-poly(p-dioxanone) (PCL-PPDX) blend in a 90:10 w:w ratio and poly(ε-caprolactone) (PCL). After achieving the standardization, a typical round-shaped chondrocyte morphology and the expression of collagen type II and aggrecan, evaluated by RT-PCR, were observed. Second-passage chondrocytes adhered effectively to these scaffolds, although cell growth at 7 days in culture was significantly less in the PCL-PPDX blend. After 3 weeks of culture on PCL-PPDX or PCL, the cells expressed collagen type II. The present study demonstrates the potential, unknown until now, of PCL-PPDX blend scaffolds in the field of cartilage tissue engineering.     

Defining conditions for covalent immobilization of angiogenic growth factors onto scaffolds for tissue engineering

Rapid vascularization of engineered tissues in vitro and in vivo remains one of the key limitations in tissue engineering. We propose that angiogenic growth factors covalently immobilized on scaffolds for tissue engineering can be used to accomplish this goal. The main objectives of this work were: (a) to derive desirable experimental conditions for the covalent immobilization of vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) on porous collagen scaffolds; and (b) to determine whether primary endothelial cells respond to these scaffolds with covalently immobilized angiogenic factors. VEGF and Ang1 were covalently immobilized onto porous collagen scaffolds, using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. To improve covalent immobilization conditions: (a) different reaction buffers [phosphate‐buffered saline (PBS), distilled water, or 2‐(N‐morpholino)ethanesulphonic acid (MES)] were used; and (b) step immobilization was compared to bulk immobilization. In step immobilization, growth factors are applied after EDC activation of the scaffold, while in bulk immobilization, reagents are simultaneously applied to the scaffold. PBS as the reaction buffer resulted in higher amounts of VEGF and Ang1 immobilized (ELISA), higher cell proliferation rates (XTT) and increased lactate metabolism compared to water and MES as the reaction buffers. Step immobilization in PBS buffer was also more effective than bulk immobilization. Immobilized growth factors resulted in higher cell proliferation and lactate metabolism compared to soluble growth factors used at comparable concentrations. Tube formation by CD31‐positive cells was also observed in collagen scaffolds with immobilized VEGF or Ang1 using H5V and primary rat aortic endothelial cells but not on control scaffolds. Copyright © 2010 John Wiley & Sons, Ltd.     

An additive manufacturing‐based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self‐assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three‐dimensional (3D) cell‐printed scaffolds using layer‐by‐layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell‐encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell‐based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL–alginate gel constructs. PCL–alginate gels containing transforming growth factor‐β (TGFβ) showed higher ECM formation. The 3D cell‐printed scaffolds of PCL–alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL–alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell‐printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.     

Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell‐free resorbable polymer scaffolds

Degeneration of intervertebral discs (IVDs) occurs frequently and is often associated with lower back pain. Recent treatment options are limited and treat the symptoms rather than regenerate the degenerated disc. Cell‐free, freeze‐dried resorbable polyglycolic acid (PGA)–hyaluronan implants were used in an ovine IVD degeneration model. The nucleus pulposus of the IVD was partially removed, endoscopically. PGA–hyaluronan implants were immersed in autologous sheep serum and implanted into the disc defect. Animals with nucleotomy only served as controls. The T2‐weighted/fat suppression sequence signal intensity index of the operated discs, as assessed by magnetic resonance imaging (MRI), showed that implantation of the PGA–hyaluronan implant improved (p = 0.0066) the MRI signal compared to controls at 6 months after surgery. Histological analysis by haematoxylin and eosin and safranin O staining showed the ingrowth of cells with typical chondrocytic morphology, even cell distribution, and extracellular matrix rich in proteoglycan. Histomorphometric analyses confirmed that the implantation of the PGA–hyaluronan scaffolds improved (p = 0.027) the formation of regenerated tissue after nucleotomy. Disc heights remained stable in discs with nucleotomy only as well as after implantation of the implant. In conclusion, implantation of cell‐free polymer‐based implants after nucleotomy induces nucleus pulposus tissue regeneration and improves disc water content in the ovine model. Copyright © 2012 John Wiley & Sons, Ltd.