Enzymatic mineralization of gellan gum hydrogel for bone tissue‐engineering applications and its enhancement by polydopamine

Interest is growing in the use of hydrogels as bone tissue‐engineering (TE) scaffolds due to advantages such as injectability and ease of incorporation of active substances such as enzymes. Hydrogels consisting of gellan gum (GG), an inexpensive calcium‐crosslinkable polysaccharide, have been applied in cartilage TE. To improve GG suitability as a material for bone TE, alkaline phosphatase (ALP), an enzyme involved in mineralization of bone by cleaving phosphate from organic phosphate, was incorporated into GG hydrogels to induce mineralization with calcium phosphate (CaP). Incorporated ALP induced formation of apatite‐like material on the submicron scale within GG gels, as shown by FTIR, SEM, EDS, XRD, ICP‐OES, TGA and von Kossa staining. Increasing ALP concentration increased amounts of CaP as well as stiffness. Mineralized GG was able to withstand sterilization by autoclaving, although stiffness decreased. In addition, mineralizability and stiffness of GG was enhanced by the incorporation of polydopamine (PDA). Furthermore, mineralization of GG led to enhanced attachment and vitality of cells in vitro while cytocompatibility of the mineralized gels was comparable to one of the most commonly used bone substitute materials. The results proved that ALP‐mediated enzymatic mineralization of GG could be enhanced by functionalization with PDA. Copyright © 2012 John Wiley & Sons, Ltd.     

RGD‐mimic polyamidoamine–montmorillonite composites with tunable stiffness as scaffolds for bone tissue‐engineering applications

This paper reports on the development of montmorillonite (MMT)‐reinforced hydrogels, based on a peptidomimetic polyamidoamine carrying guanidine pendants (AGMA1), as substrates for the osteo‐induction of osteoblast precursor cells. AGMA1 hydrogels of various degrees of crosslinking responded favourably to MMT reinforcement, giving rise to composite hydrogels with shear storage modulus G′, when fully swollen in water, up to 200 kPa, i.e. 20 times higher than the virgin hydrogels and of the same order or higher than other hydrogel‐based composites proposed for orthopaedic applications. This significant improvement was ascribed to the effective interpenetration between the polymer matrix and the inorganic filler. AGMA1–MMT hydrogels, when evaluated as scaffolds for the osteogenic differentiation of mouse calvaria‐derived pre‐osteoblastic MC3T3‐E1 cells, proved able to support cell adhesion and proliferation and clearly induced differentiation towards the osteoblastic phenotype, as indicated by different markers. In addition, AGMA1–MMT hydrogels proved completely degradable in aqueous media at pH 7.4 and did not provide any evidence of cytotoxicity. The experimental evidence suggests that AGMA1–MMT composites definitely warrant potential as scaffolds for osteoblast culture and bone grafts. Copyright © 2016 John Wiley & Sons, Ltd.     

Anti‐angiogenic potential of VEGF blocker dendron loaded on to gellan gum hydrogels for tissue engineering applications

Damage of non‐vascularised tissues such as cartilage and cornea can result in healing processes accompanied by a non‐physiological angiogenesis. Peptidic aptamers have recently been reported to block the vascular endothelial growth factor (VEGF). However, the therapeutic applications of these aptamers are limited due to their short half‐life in vivo. In this work, an enhanced stability and bioavailability of a known VEGF blocker aptamer sequence (WHLPFKC) was pursued through its tethering of molecular scaffolds based on hyperbranched peptides, the poly(?‐lysine) dendrons, bearing three branching generations. The proposed design allowed simultaneous and orderly‐spaced exposure of 16 aptamers per dendrimer to the surrounding biological microenvironent, as well as a relatively hydrophobic core based on di‐phenylalanine aiming to promote an hydrophobic interaction with the hydrophobic moieties of ionically crosslinked methacrylated gellan gum (iGG‐MA) hydrogels. The VEGF blocker dendrons were entrapped in iGG‐MA hydrogels, and their capacity to prevent endothelial cell sprouting was assessed qualitatively and quantitatively using 3D in vitro models and the in vivo chick chorioallantoic membrane assay. The data demonstrate that at nanoscale concentrations, the dendronised structures were able to enhance control of the biological actvity of WHLPFKC at the material/tissue interface and hence the anti‐angiogenic capacity of iGG‐MA hydrogels not only preventing blood vessel invasion, but also inducing their regression at the tissue/iGG‐MA interface. The in ovo study confirmed that iGG‐MA functionalised with the dendron VEGF blockers do inhibit angiogenesis by controlling both size and ramifications of blood vessels in the proximity of the implanted gel surface. Copyright © 2016 John Wiley & Sons, Ltd.     

Generation of composites for bone tissue‐engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means

Mineralization of hydrogels, desirable for bone regeneration applications, may be achieved enzymatically by incorporation of alkaline phosphatase (ALP). ALP‐loaded gellan gum (GG) hydrogels were mineralized by incubation in mineralization media containing calcium and/or magnesium glycerophosphate (CaGP, MgGP). Mineralization media with CaGP:MgGP concentrations 0.1:0, 0.075:0.025, 0.05:0.05, 0.025:0.075 and 0:0.1 (all values mol/dm³, denoted A, B, C, D and E, respectively) were compared. Mineral formation was confirmed by IR and Raman, SEM, ICP‐OES, XRD, TEM, SAED, TGA and increases in the the mass fraction of the hydrogel not consisting of water. Ca was incorporated into mineral to a greater extent than Mg in samples mineralized in media A–D. Mg content and amorphicity of mineral formed increased in the order A < B < C < D. Mineral formed in media A and B was calcium‐deficient hydroxyapatite (CDHA). Mineral formed in medium C was a combination of CDHA and an amorphous phase. Mineral formed in medium D was an amorphous phase. Mineral formed in medium E was a combination of crystalline and amorphous MgP. Young's moduli and storage moduli decreased in dependence of mineralization medium in the order A > B > C > D, but were significantly higher for samples mineralized in medium E. The attachment and vitality of osteoblastic MC3T3‐E1 cells were higher on samples mineralized in media B–E (containing Mg) than in those mineralized in medium A (not containing Mg). All samples underwent degradation and supported the adhesion of RAW 264.7 monocytic cells, and samples mineralized in media A and B supported osteoclast‐like cell formation. Copyright © 2014 John Wiley & Sons, Ltd.     

Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue‐engineering strategies; translational issues

Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l ‐lactic acid)–poly(ε‐caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow‐derived skeletal stem cells (SSCs) were implanted into mid‐shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro‐CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical‐sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale‐up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale‐up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

Gelatin‐based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF‐controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4‐D6P2T) by assessing VEGFR‐2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF‐releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin‐based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

An in vitro expansion score for tissue‐engineering applications with human bone marrow‐derived mesenchymal stem cells

Human bone marrow‐derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue‐engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony‐forming unit (CFU) assay, population doubling time (PDT), senescence‐associated β‐galactosidase (SA‐β‐Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA‐β‐Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late‐passage cells than in early‐passage cells. Early‐passage cells underwent efficient osteogenic differentiation, with mid‐passage cells performing best in chondrogenic differentiation. Late‐passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.     

Biocompatibility of two model elastin‐like recombinamer‐based hydrogels formed through physical or chemical cross‐linking for various applications in tissue engineering and regenerative medicine

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein‐based ones, such as elastin‐like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix‐like hydrogels through either physical or chemical cross‐linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase‐expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR‐based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL‐1β, IL‐4, IL‐6, and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine‐related applications.     

Cell bricks‐enriched platelet‐rich plasma gel for injectable cartilage engineering – an in vivo experiment in nude mice

Clinical application of platelet‐rich plasma (PRP)‐based injectable tissue engineering is limited by weak mechanical properties and a rapid fibrinolytic rate. We proposed a new strategy, a cell bricks‐stabilized PRP injectable system, to engineer and regenerate cartilage with stable morphology and structure in vivo. Chondrocytes from the auricular cartilage of rabbits were isolated and cultured to form cell bricks (fragmented cell sheet) or cell expansions. Fifteen nude mice were divided evenly (n = 5) into cells–PRP (C‐P), cell bricks–PRP (CB‐P) and cell bricks–cells–PRP (CB‐C‐P) groups. Cells, cell bricks or a cell bricks/cells mixture were suspended in PRP and were injected subcutaneously in animals. After 8 weeks, all the constructs were replaced by white resilient tissue; however, specimens from the CB‐P and CB‐C‐P groups were well maintained in shape, while the C‐P group appeared distorted, with a compressed outline. Histologically, all groups presented lacuna‐like structures, glycosaminoglycan‐enriched matrices and positive immunostaining of collagen type II. Different from the uniform structure presented in CB‐C‐P samples, CB‐P presented interrupted, island‐like chondrogenesis and contracted structure; fibrous interruption was shown in the C‐P group. The highest percentage of matrix was presented in CB‐C‐P samples. Collagen and sGAG quantification confirmed that the CB‐C‐P constructs had statistically higher amounts than the C‐P and CB‐P groups; statistical differences were also found among the groups in terms of biomechanical properties and gene expression. We concluded that cell bricks‐enriched PRP gel sufficiently enhanced the morphological stability of the constructs, maintained chondrocyte phenotypes and favoured chondrogenesis in vivo, which suggests that such an injectable, completely biological system is a suitable cell carrier for cell‐based cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.     

Pooled thrombin‐activated platelet‐rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin‐activated, pooled, platelet‐rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9‐fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7‐fold increase) in constructs cultured with tPRP. Staining for human‐specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose‐derived cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Micro‐computed tomography for implantation site imaging during in situ oesophagus tissue engineering in a live small animal model

For tissue engineering of gastrointestinal organs, in situ implantation of constructs in the omentum is performed to utilize the body as a bioreactor for tissue generation. In this approach, constructs are fabricated into tubes, using stents, and implanted in the omentum to induce vascularization. In order to evaluate the constructs and its environment during the period of in situ tissue engineering in the rat model, micro‐computed tomography imaging was performed. Imaging using micro‐computed tomography was useful in localization of the position of the construct, evaluation of implant site tissue, degree of peripheral inflammation to neighbouring tissues and migration of the implanted construct. Images also enable the estimation of the dimensions of the construct and imaging of cyst formations or fluid accumulations on the luminal side of the tubular construct or ascites formation. Since micro‐computed tomography is a non‐invasive method, it can be repeated for evaluation of implanted constructs if in situ tissue engineering is performed over longer periods. Copyright © 2009 John Wiley & Sons, Ltd.     

Human endothelial colony‐forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold‐based tissue engineering

One of the major challenges in tissue engineering is to supply larger three‐dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)‐derived endothelial colony‐forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue‐culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo‐expanded ECFCs are a novel, very potent cell source for scaffold‐based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Local delivery of allogeneic bone marrow and adipose tissue‐derived mesenchymal stromal cells for cutaneous wound healing in a porcine model

Wound healing remains a major challenge in modern medicine. Bone marrow‐ (BM) and adipose tissue‐ (AT) derived mesenchymal stromal/stem cells (MSCs) are of great interest for tissue reconstruction due to their unique immunological properties and regenerative potential. The purpose of this study was to characterize BM and AT‐MSCs and evaluate their effect when administered in a porcine wound model. MSCs were derived from male Göttingen Minipigs and characterized according to established criteria. Allogeneic BM‐ or AT‐MSCs were administered intradermally (1 x 10⁶ cells) into partial‐thickness wounds created on female animals, and covered with Vaseline® gauze or fibrin in a randomized pattern. Animals were euthanized at 7, 10, 14 and 21 days. Tissues were analyzed visually for healing and by microscopic examination for epidermal development and remodelling. Polymerase chain reaction (PCR) was used to detect the presence of male DNA in the specimens. All wounds were healed by 14 days. MSC‐injected wounds were associated with improved appearance and faster re‐epithelialization compared to saline controls. Evaluation of rete ridge depth and architecture showed that MSC treatment promoted a faster rate of epidermal maturation. Male DNA was detected in all samples at days 7 and 10, suggesting the presence of MSCs. We showed the safety, feasibility and potential efficacy of local injection of allogeneic BM‐ and AT‐MSCs for treatment of wounds in a preclinical model. Our data in this large animal model support the potential use of BM‐ and AT‐MSC for treatment of cutaneous wounds through modulation of healing and epithelialization. Copyright © 2013 John Wiley & Sons, Ltd.     

The ideal way to design clinical trials and establishment of evidence for human cellular and tissue‐based products in Japan

In Japan, the Pharmaceuticals and Medical Devices (PMD) Act established in 2014 included an additional chapter dedicated to frameworks for human cellular and tissue‐based products. To further evaluate the product considered to have “likely to predict efficacy” at the time of receiving “conditional and time‐limited marketing authorization,” a system has been introduced to determine whether the product is eligible for “full marketing authorization” through statistical “confirmation of the efficacy” in a postmarketing surveillance study using a registry. A movement similar to this regulation has been seen among Western nations. For example, in the United States, Regenerative Medicine Advanced Therapy Designation was introduced in 2017 as a provision of the 21st Century Cures Act. This is similar to Japan's conditional and time‐limited marketing authorization, which presumes efficacy of a product based on surrogate endpoints in life‐threatening diseases. It is true that the current study design has limitations, and study designs that are beyond our imagination should be developed in the future.