Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Functional cell phenotype induction with TGF-ß1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor ß1 (TGF-ß1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-ß1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-ß1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-ß1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-ß1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-ß1-containing collagen-PU constructs can induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

Chondrogenic potential of IL-10 in mechanically injured cartilage and cellularized collagen ACI grafts.

OBJECTIVE: The application of adjunctive mediators in Autologous chondrocyte implantation (ACI) techniques might be useful for improving the dedifferentiated chondrocyte phenotype, to support neocartilage formation and inhibit post-traumatic cartilage destruction. In this study we examined if (a) interleukin 10 treatment can cause chondrogenic phenotype stabilization and matrix preservation in mechanically injured cartilage and if (b) IL-10 can promote chondrogenesis in a clinically applied collagen scaffold for ACI treatment. MATERIALS AND METHODS: For (a) bovine articular cartilage was harvested, subjected to an axial unconfined injury and treated with bovine IL-10 (1-10,000 pg/ng/ml). For (b) a post-operatively remaining ACI graft was treated with human IL-10. Expression levels of type I/II/X collagen, SOX9 and aggrecan were measured by qPCR (a,b). After 3 weeks cell death was analyzed (nuclear blebbing and TUNEL assay) and matrix composition was determined by GAG measurements and immunohistochemistry (aggrecan, type I/II collagen, hyaluronic acid). STATISTICS: One way ANOVA analysis with Bonferroni's correction. RESULTS: (a) IL-10 stabilized the chondrogenic phenotype after injurious compression and preserved matrix integrity. This was indicated by elevated expression of chondrogenic markers COL2A1, ACAN, SOX9, while COL1A1 and COL10A1 were reduced. An increased GAG content paralleled this and histological staining of type 2 collagen, aggrecan and toluidine blue were enhanced after 3 weeks. (b) IL-10 [100 pg/ml] improved the chondrogenic differentiation of human chondrocytes, which was accompanied by cartilaginous matrix formation after 3 weeks of incubation. CONCLUSION: Interleukin-10 is a versatile adjuvant candidate to control the post-injurious environment in cartilage defects and promote chondrogenesis in ACI grafts.

Thermoreversible hyaluronan-hydrogel and autologous nucleus pulposus cell delivery regenerates human intervertebral discs in an ex vivo, physiological organ culture model.

Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1ρ magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1ρ values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture set-up, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.

Local application of a gentamicin-loaded thermo-responsive hydrogel allows for fracture healing upon clearance of a high Staphylococcus aureus load in a rabbit model.

Antibiotic-loaded biomaterials (ALBs) have emerged as a potential useful adjunctive antimicrobial measure for the prevention of infection in open fracture care. A biodegradable thermo-responsive poly(N-isopropylacrylamide) grafted hyaluronic acid (HApN) hydrogel loaded with gentamicin has recently been shown to prevent implant-related infection in a rabbit osteosynthesis model. The primary aim of this study was to determine the influence of this HApN hydrogel on bone healing at an early stage (4 weeks). A rabbit humeral osteotomy model with plating osteosynthesis was used to compare fracture healing in rabbits receiving the hydrogel as compared with control animals. The secondary aim was to observe fracture healing in groups treated with and without antibiotic-loaded hydrogel in the presence of bacterial contamination. In all groups, outcome measures were mechanical stability and histological score, with additional quantitative bacteriology in the inoculated groups. Application of the HApN hydrogel in non-inoculated rabbits did not significantly influence humeral stiffness or histological scores for fracture healing in comparison to controls. In the inoculated groups, animals receiving the bacterial inoculum without hydrogel were culture-positive at euthanasia and found to display lower humeral stiffness values and higher histopathological scores for bacterial presence in comparison with equivalents receiving the gentamicin-loaded HApN hydrogel, which were also infection-free. In summary, our data showed that HApN was an effective antibiotic carrier that did not affect fracture healing. This data supported its suitability for application in fracture care. Addition of osteopromotive compounds could provide further support for accelerating fracture healing in addition to successful infection prophylaxis.

Effects of macrophage-activating lipopeptide-2 (MALP-2) on the vascularisation of implanted polyurethane scaffolds seeded with microvascular fragments.

The seeding of scaffolds with adipose tissue-derived microvascular fragments represents a promising strategy to establish a sufficient blood supply in tissue constructs. Herein, we analysed whether a single application of macrophage-activating lipopeptide-2 (MALP-2) at the implantation site further improves the early vascularisation of such microvessel-seeded constructs. Microvascular fragments were isolated from epididymal fat pads of C57BL/6 mice. The fragments were seeded on polyurethane scaffolds, which were implanted into mouse dorsal skinfold chambers exposed to MALP-2 or vehicle (control). The inflammatory host tissue response and the vascularisation of the scaffolds were analysed using intravital fluorescence microscopy, histology and immunohistochemistry. We found that the numbers of microvascular adherent leukocytes were significantly increased in MALP-2-treated chambers during the first 3 days after scaffold implantation when compared to controls. This temporary inflammation resulted in an improved vascularisation of the host tissue surrounding the implants, as indicated by a higher density of CD31-positive microvessels at day 14. However, the MALP-2-exposed scaffolds themselves presented with a lower functional microvessel density in their centre. In addition, in vitro analyses revealed that MALP-2 promotes apoptotic cell death of endothelial and perivascular cells in isolated microvascular fragments. Hence, despite the beneficial pro-angiogenic properties of MALP-2 at the implantation site, the herein evaluated approach may not be recommended to improve the vascularisation capacity of microvascular fragments in tissue engineering applications.

Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor.

Low back pain originating from intervertebral disc (IVD) degeneration affects the quality of life for millions of people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivo models of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. A bioreactor was developed that facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.3 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility of tissue repair strategies with cell supplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.

Innovating in the medical device industry - challenges & opportunities ESB 2015 translational research symposium.

The European Society for Biomaterials 2015 Translational Research Symposium focused on 'Innovating in the Medical Device Industry - Challenges & Opportunities' from different perspectives, i.e., from a non-profit research organisation to a syndicate of small and medium-sized companies and large companies. Lecturers from regulatory consultants, industry and research institutions described the innovation process and regulatory processes (e.g., 510K, PMA, combination product) towards market approval. The aim of the present article is to summarise and explain the main statements made during the symposium, in terms of challenges and opportunities for medical device industries, in a constantly changing customer and regulatory environment.

Pre-cultivation of adipose tissue-derived microvascular fragments in porous scaffolds does not improve their in vivo vascularisation potential.

Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFP-positive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, non-cultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.

Progressing innovation in biomaterials. From the bench to the bed of patients.

A Translational Research Symposium was organized at the 2014 annual meeting of the European society for biomaterials. This brought together leading Tier one companies in clinical biomaterials and medical device markets, small and medium enterprises and entrepreneurial academics who shared their experiences on taking biomaterials technologies to commercial endpoints, in the clinics. The symposium focused on "Progressing Innovation in Biomaterials. From the Bench to the Bed of Patients". The aim of the present document is to illustrate the content of the symposium and to highlight the key lessons from selected lectures.

Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity.

Adipose tissue-derived microvascular fragments are promising vascularisation units for applications in the field of tissue engineering. Elderly patients are the major future target population of such applications due to an increasing human life expectancy. Therefore, we herein investigated the effect of aging on the fragments' vascularisation capacity. Microvascular fragments were isolated from epididymal fat pads of adult (8 months) and aged (16 months) C57BL/6 donor mice. These fragments were seeded onto porous polyurethane scaffolds, which were implanted into dorsal skinfold chambers to study their vascularisation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with fragments from aged donors exhibited a significantly lower functional microvessel density and intravascular blood flow velocity. This was associated with an impaired vessel maturation, as indicated by vessel wall irregularities, constantly elevated diameters and a lower fraction of CD31/α-smooth muscle actin double positive microvessels in the implants' border and centre zones. Additional in vitro analyses revealed that microvascular fragments from adult and aged donors do not differ in their stem cell content as well as in their release of angiogenic growth factors, survival and proliferative activity under hypoxic conditions. However, fragments from aged donors exhibit a significantly lower number of matrix metalloproteinase -9-positive perivascular cells. Taken together, these findings demonstrate that aging is a crucial determinant for the vascularisation capacity of isolated microvascular fragments.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in a simulated osteochondral environment is hydrogel dependent.

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.

Challenges and strategies in the repair of ruptured annulus fibrosus.

Lumbar discectomy is the surgical procedure most frequently performed for patients suffering from low back pain and sciatica. Disc herniation as a consequence of degenerative or traumatic processes is commonly encountered as the underlying cause for the painful condition. While discectomy provides favourable outcome in a majority of cases, there are conditions where unmet requirements exist in terms of treatment, such as large disc protrusions with minimal disc degeneration; in these cases, the high rate of recurrent disc herniation after discectomy is a prevalent problem. An effective biological annular repair could improve the surgical outcome in patients with contained disc herniations but otherwise minor degenerative changes. An attractive approach is a tissue-engineered implant that will enable/stimulate the repair of the ruptured annulus. The strategy is to develop three-dimensional scaffolds and activate them by seeding cells or by incorporating molecular signals that enable new matrix synthesis at the defect site, while the biomaterial provides immediate closure of the defect and maintains the mechanical properties of the disc. This review is structured into (1) introduction, (2) clinical problems, current treatment options and needs, (3) biomechanical demands, (4) cellular and extracellular components, (5) biomaterials for delivery, scaffolding and support, (6) pre-clinical models for evaluation of newly developed cell- and material-based therapies, and (7) conclusions. This article highlights that an interdisciplinary approach is necessary for successful development of new clinical methods for annulus fibrosus repair. This will benefit from a close collaboration between research groups with expertise in all areas addressed in this review.

Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments.

In tissue engineering, the generation of tissue constructs comprising preformed microvessels is a promising strategy to guarantee their adequate vascularisation after implantation. Herein, we analysed whether this may be achieved by seeding porous scaffolds with adipose tissue-derived microvascular fragments. Green fluorescent protein (GFP)-positive microvascular fragments were isolated by enzymatic digestion from epididymal fat pads of male C57BL/6-TgN(ACTB-EGFP)1Osb/J mice. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were seeded with these fragments and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study inosculation and vascularisation of the implants by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Empty scaffolds served as controls. Vital microvascular fragments could be isolated from adipose tissue and seeded onto the scaffolds under dynamic pressure conditions. In the dorsal skinfold chamber, the fragments survived and exhibited a high angiogenic activity, resulting in the formation of GFP-positive microvascular networks within the implants. These networks developed interconnections to the host microvasculature, resulting in a significantly increased functional microvessel density at day 10 and 14 after implantation when compared to controls. Immunohistochemical analyses of vessel-seeded scaffolds revealed that >90 % of the microvessels in the implants' centre and ~60 % of microvessels in the surrounding host tissue were GFP-positive. This indicates that the scaffolds primarily vascularised by external inosculation. These novel findings demonstrate that the vascularisation of implanted porous scaffolds can be improved by incorporation of microvascular fragments. Accordingly, this approach may markedly contribute to the success of future tissue engineering applications in clinical practice.

The production and application of bacterial exopolysaccharides as biomaterials for bone regeneration.

Bacterial exopolysaccharides (EPS) are water-soluble polymers consisting of repeating sugar moieties that serve a wide range of functions for the bacterial species that produce them. Their functions include biofilm matrix constituent, nutrient retention, protection from environmental threats and even pathogenicity. EPS have also been exploited for use in various applications in the biomedical field: most notably as viscosupplements, drug delivery vehicles and in tissue engineering constructs. The use of EPS in bone tissue engineering has increased in recent years due to the wide range of compounds available, low cost, and ease of production on an industrial scale. This review discusses the extraction and purification methods employed to produce bacterial EPS. A particular focus is on bone-related tissue engineering applications where EPS is the primary active agent, or as a scaffold matrix, as well as a carrier for osteopromotive agents.

The waves that make the pattern: a review on acoustic manipulation in biomedical research.

Novel approaches, combining technology, biomaterial design, and cutting-edge cell culture, have been increasingly considered to advance the field of tissue engineering and regenerative medicine. Within this context, acoustic manipulation to remotely control spatial cellular organization within a carrier matrix has arisen as a particularly promising method during the last decade. Acoustic or sound-induced manipulation takes advantage of hydrodynamic forces exerted on systems of particles within a liquid medium by standing waves. Inorganic or organic particles, cells, or organoids assemble within the nodes of the standing wave, creating distinct patterns in response to the applied frequency and amplitude. Acoustic manipulation has advanced from micro- or nanoparticle arrangement in 2D to the assembly of multiple cell types or organoids into highly complex in vitro tissues. In this review, we discuss the past research achievements in the field of acoustic manipulation with particular emphasis on biomedical application. We survey microfluidic, open chamber, and high throughput devices for their applicability to arrange non-living and living units in buffer or hydrogels. We also investigate the challenges arising from different methods, and their prospects to gain a deeper understanding of in vitro tissue formation and application in the field of biomedical engineering.

Promoting external inosculation of prevascularised tissue constructs by pre-cultivation in an angiogenic extracellular matrix.

The engineering of preformed microvessels offers the promising opportunity to rapidly vascularise implanted tissue constructs by the process of inosculation. Herein, we analyzed whether this process may further be accelerated by cultivation of prevascularised tissue constructs in Matrigel before implantation. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were implanted into the flank of FVB/N-TgN (Tie2/GFP) 287 Sato mice to allow the ingrowth of a granulation tissue with green fluorescent protein (GFP)-positive blood vessels. After harvesting, these prevascularised constructs were then transferred into dorsal skinfold chambers of FVB/N recipient mice to study the process of inosculation. The constructs were implanted directly after embedding in Matrigel or after 3 days of cultivation in the extracellular matrix. Matrigel-free constructs served as control. Cultivation in Matrigel resulted in the outgrowth of CD31/GFP-positive vascular sprouts. Vascularisation of these constructs was markedly improved when compared to the other two groups, as indicated by a significantly elevated functional microvessel density between days 6 to 14 after implantation into the dorsal skinfold chamber. This was associated with an increased number of GFP-positive blood vessels growing into the surrounding host tissue. Thus, the blood supply to prevascularised tissue constructs can be accelerated by their pre-cultivation in an angiogenic extracellular matrix, promoting external inosculation of the preformed microvascular networks with the host microvasculature.

Efficacy of antimicrobial agents delivered to hernia meshes using an adaptable thermo-responsive hyaluronic acid-based coating.

PURPOSE: Mesh-related infection is a critical outcome for patients with hernia defect stabilized with synthetic or biological meshes. Even though bioactive meshes loaded with antibiotics or antiseptics are slowly emerging in the market, the available solutions still lack versatility. Here, we proposed a polymer solution, i.e., hyaluronic acid-poly(N-isopropylacrylamide) (HApN), which forms a hydrogel to be used as coating for meshes only when it reaches body temperature. METHODS: We assessed how the gelation of HApN was influenced by the incorporation of different antibiotic and antiseptic formulations, and how this gel can be used to coat several mesh types. The impact of the coating on the elastic behavior of a macroporous mesh was tested under cyclic elongation condition. Finally, we selected two different coating formulations, one based on antibiotics (gentamicin + rifampicin) and one based on antiseptic (chlorhexidine) and tested in vitro their antimicrobial efficacies. RESULTS: HApN can be used as carrier for different antimicrobial agents, without having a strong influence on its gelation behavior. Porous or dense meshes can be coated with this polymer, even though the stability was not optimal on macroporous meshes such as Optilene when pores are too large. HApN loaded with drugs inhibited in vitro the growth of several Gram-positive and Gram-negative bacteria. CONCLUSION: Compared to the available technologies developed to endow meshes with antibacterial activity, the proposed HApN offers further versatility with potential to prevent mesh-related infection in hernioplasty.

Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell migration and alignment.

Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes and gradients of composition and biological cues. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the micron and the molecular level. Herein, we introduce a bioink containing the tyramine derivative of hyaluronan (HA; henceforth known as THA) and collagen (Col) type 1. In this bioink, similar to connective tissues, Col is present in the fibrillar form, and HA functions as a viscoelastic space filler. THA was enzymatically cross-linked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. The THA-Col composite displayed synergistic properties in terms of storage modulus and shear thinning, translating into good printability. Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second-harmonic generation. Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on human bone marrow-derived mesenchymal stromal cell (hMSC) migration. Anisotropic HA-Col showed cell-instructive properties modulating hMSC adhesion, morphology, and migration from micropellets stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded in aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and ECM production, hMSCs embedded in the isotropic bioink displayed chondrogenic differentiation comparable with standard pellet culture by means of proteoglycan production (safranin O staining and proteoglycan quantification). The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.

Hyaluronic acid as a bioink for extrusion-based 3D printing.

Biofabrication is enriching the tissue engineering field with new ways of producing structurally organized complex tissues. Among the numerous bioinks under investigation, hyaluronic acid (HA) and its derivatives stand out for their biological relevance, cytocompatibility, shear-thinning properties, and potential to fine-tune the desired properties with chemical modification. In this paper, we review the recent advances on bioinks containing HA. The available literature is presented based on subjects including the rheological properties in connection with printability, the chemical strategies for endowing HA with the desired properties, the clinical application, the most advanced preclinical studies, the advantages and limitations in comparison with similar biopolymer-based bioinks, and future perspectives.