Engineering complex tissue-like microenvironments with biomaterials and biofabrication.

Advances in tissue engineering for both system modeling and organ regeneration depend on embracing and recapitulating the target tissue's functional and structural complexity. Microenvironmental features such as anisotropy, heterogeneity, and other biochemical and mechanical spatiotemporal cues are essential in regulating tissue development and function. Novel biofabrication strategies and innovative biomaterial design have emerged as promising tools to better reproduce such features. These facilitate a transition towards high-fidelity biomimetic structures, offering opportunities for a deeper understanding of tissue function and the development of superior therapies. In this review, we explore some of the key structural and compositional aspects of tissues, lay out how to achieve similar outcomes with current fabrication strategies, and identify the main challenges and promising avenues for future research.

Optimizing the physical properties of collagen/hyaluronan hydrogels by inhibition of polyionic complexes formation at pH close to the collagen isoelectric point.

Collagen/hyaluronan hydrogels with physical properties well suited for biomedical applications are challenging to synthesize due to the formation of polyionic complexes (PICs). A systematic physicochemical study was thus performed to determine novel conditions to inhibit the formation of collagen/hyaluronan PICs and obtain composite hydrogels with high physical properties. Using a range of pH from 1 to 5.5 and the addition of NaCl, type I collagen and tyramine-substituted hyaluronic acid (THA) solutions were mixed and analyzed by cryo-scanning electron microscopy and ATR-FTIR. PIC formation was inhibited at pH 1 without salt and at pH 2.5 and 5.5 in the presence of 400 mM NaCl. Interestingly, collagen fibrils were observed in solution at pH 5.5 before mixing with THA. After collagen gelling by pH increase, a homogeneous hydrogel consisting of collagen fibrils was only observed when PICs were inhibited. Then, the THA gelling performed by photo-crosslinking increased the rheological properties by four when hydrogels were formed with collagen/THA mixtures at pH 1 or 5.5 with salt. Taken together, these results show that a pH of 5.5, close to the collagen isoelectric point, enables the formation of collagen fibrils in solution, inhibits the PICs formation, and allows the formation of homogenous collagen/THA composite hydrogels compatible with cell survival.

Alginate microbeads and hydrogels delivering meropenem and bacteriophages to treat Pseudomonas aeruginosa fracture-related infections.

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9-30 and MK.R3-15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future.

Combining biomimetic collagen/hyaluronan hydrogels with discogenic growth factors promotes mesenchymal stroma cell differentiation into Nucleus Pulposus like cells.

Based on stem cell injection into degenerated Nucleus Pulposus (NP), novel treatments for intervertebral disc (IVD) regeneration were disappointing because of cell leakage or inappropriate cell differentiation. In this study, we hypothesized that mesenchymal stromal cells encapsulated within injectable hydrogels possessing adequate physico-chemical properties would differentiate into NP like cells. Composite hydrogels consisting of type I collagen and tyramine-substituted hyaluronic acid (THA) were prepared to mimic the NP physico-chemical properties. Human bone marrow derived mesenchymal stromal cells (BM-MSCs) were encapsulated within hydrogels and cultivated in proliferation medium (supplemented with 10% fetal bovine serum) or differentiation medium (supplemented with GDF5 and TGFß1) over 28 days. Unlike pure collagen, collagen/THA composite hydrogels were stable over 28 days in culture. In proliferation medium, the cell viability within pure collagen hydrogels was high, whereas that in composite and pure THA hydrogels was lower due to the weaker cell adhesion. Nonetheless, BM-MSCs proliferated in all hydrogels. In composite hydrogels, cells exhibited a rounded morphology similar to NP cells. The differentiation medium did not impact the hydrogel stability and cell morphology but negatively impacted the cell viability in pure collagen hydrogels. A high THA content within hydrogels promoted the gene expression of NP markers such as collagen II, aggrecan, SOX9 and cytokeratin 18 at day 28. The differentiation medium potentialized this effect with an earlier and higher expression of these NP markers. Taken together, these results show that the physico-chemical properties of collagen/THA composite hydrogels and GDF5/TGFß1 act in synergy to promote the differentiation of BM-MSCs into NP like cells.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69 kPa to 2.24 kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Development of a hyaluronic acid-collagen bioink for shear-induced fibers and cells alignment.

Human tissues are characterized by complex composition and cellular and extracellular matrix (ECM) organization at microscopic level. In most of human tissues, cells and ECM show an anisotropic arrangement, which confers them specific properties.In vitro, the ability to closely mimic this complexity is limited. However, in the last years, extrusion bioprinting showed a certain potential for aligning cells and biomolecules, due to the application of shear stress during the bio-fabrication process. In this work, we propose a strategy to combine collagen (col) with tyramine-modified hyaluronic acid (THA) to obtain a printable col-THA bioink for extrusion bioprinting, solely-based on natural-derived components. Collagen fibers formation within the hybrid hydrogel, as well as collagen distribution and spatial organization before and after printing, were studied. For the validation of the biological outcome, fibroblasts were selected as cellular model and embedded in the col-THA matrix. Cell metabolic activity and cell viability, as well as cell distribution and alignment, were studied in the bioink before and after bioprinting. Results demonstrated successful collagen fibers formation within the bioink, as well as collagen anisotropic alignment along the printing direction. Furthermore, results revealed suitable biological properties, with a slightly reduced metabolic activity at day 1, fully recovered within the first 3 d post-cell embedding. Finally, results showed fibroblasts elongation and alignment along the bioprinting direction. Altogether, results validated the potential to obtain collagen-based bioprinted constructs, with both cellular and ECM anisotropy, without detrimental effects of the fabrication process on the biological outcome. This bioink can be potentially used for a wide range of applications in tissue engineering and regenerative medicine in which anisotropy is required.

Modulating design parameters to drive cell invasion into hydrogels for osteochondral tissue formation.

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p â€‹= â€‹0.390) and led to a higher migration score ex vivo (p â€‹< â€‹0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p â€‹= â€‹0.031) and ex vivo (p â€‹< â€‹0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p â€‹< â€‹0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used.The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application.

3D Printing of Extracellular Matrix-Based Multicomponent, All-Natural, Highly Elastic, and Functional Materials toward Vascular Tissue Engineering.

3D printing offers an exciting opportunity to fabricate biological constructs with specific geometries, clinically relevant sizes, and functions for biomedical applications. However, successful application of 3D printing is limited by the narrow range of printable and bio-instructive materials. Multicomponent hydrogel bioinks present unique opportunities to create bio-instructive materials able to display high structural fidelity and fulfill the mechanical and functional requirements for in situ tissue engineering. Herein, 3D printable and perfusable multicomponent hydrogel constructs with high elasticity, self-recovery properties, excellent hydrodynamic performance, and improved bioactivity are reported. The materials' design strategy integrates fast gelation kinetics of sodium alginate (Alg), in situ crosslinking of tyramine-modified hyaluronic acid (HAT), and temperature-dependent self-assembly and biological functions of decellularized aorta (dAECM). Using extrusion-based printing approach, the capability to print the multicomponent hydrogel bioinks with high precision into a well-defined vascular constructs able to withstand flow and repetitive cyclic compressive loading, is demonstrated. Both in vitro and pre-clinical models are used to show the pro-angiogenic and anti-inflammatory properties of the multicomponent vascular constructs. This study presents a strategy to create new bioink whose functional properties are greater than the sum of their components and with potential applications in vascular tissue engineering and regenerative medicine.

Swelling-Dependent Shape-Based Transformation of a Human Mesenchymal Stromal Cells-Laden 4D Bioprinted Construct for Cartilage Tissue Engineering.

3D bioprinting is usually implemented on flat surfaces, posing serious limitations in the fabrication of multilayered curved constructs. 4D bioprinting, combining 3D bioprinting with time-dependent stimuli-induced transformation, enables the fabrication of shape-changing constructs. Here, a 4D biofabrication method is reported for cartilage engineering based on the differential swelling of a smart multi-material system made from two hydrogel-based materials: hyaluronan and alginate. Two ink formulations are used: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Both inks have similar elastic, shear-thinning, and printability behavior. The inks are 3D printed into a bilayered scaffold before triggering the shape-change by using liquid immersion as stimulus. In time (4D), the differential swelling between the two zones leads to the scaffold's self-bending. Different designs are made to tune the radius of curvature and shape. A bioprinted formulation of AHAT and human bone marrow cells demonstrates high cell viability. After 28 days in chondrogenic medium, the curvature is clearly present while cartilage-like matrix production is visible on histology. A proof-of-concept of the recently emerged technology of 4D bioprinting with a specific application for the design of curved structures potentially mimicking the curvature and multilayer cellular nature of native cartilage is demonstrated.

A culture model to analyze the acute biomaterial-dependent reaction of human primary neutrophils in vitro.

Neutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.

Fabrication of Collagen-Hyaluronic Acid Cryogels by Directional Freezing Mimicking Cartilage Arcade-like Structure.

The internal architecture of tissue-like constructs is fundamental to their structural and biological functions. Here, we introduce a simple and robust method to fabricate cryogels based on derivatized extracellular matrix (ECM) macromolecules with porosity arranged according to the typical Benninghoff zonal architecture of articular cartilage. To obtain this arcade-like structure, the technique used the growth of ice crystals from copper pins at cryogenic temperatures. The directional cryogel formation enabled the organized growth of ice crystals over a large distance (>4 mm). The compositional properties were achieved by forming double networks (DNs) of hyaluronic acid and collagen derivatives (MeHA and CollGTA, respectively), which also served to improve the mechanical properties of the otherwise weak collagen scaffolds. Compositionally biomimetic and more resilient MeHA-CollGTA DNs (Young's modulus ≈ 200 kilopascals) were therefore produced. The technique presented expands the fabrication methods available for providing ECM macromolecules with architectural elements mimicking cartilage complexity.

Tuning the Cell-Adhesive Properties of Two-Component Hybrid Hydrogels to Modulate Cancer Cell Behavior, Metastasis, and Death Pathways.

This work presents a polysaccharide and protein-based two-component hybrid hydrogel integrating the cell-adhesive gelatin-tyramine (G-Tyr) and nonadhesive hyaluronic acid-tyramine (HA-Tyr) through enzyme-mediated oxidative coupling reaction. The resulting HA-Tyr/G-Tyr hydrogel reflects the precise chemical and mechanical features of the cancer extracellular matrix and is able to tune cancer cell adhesion upon switching the component ratio. The cells form quasi-spheroids on HA-Tyr rich hydrogels, while they tend to form an invasive monolayer culture on G-Tyr rich hydrogels. The metastatic genotype of colorectal adenocarcinoma cells (HT-29) increases on G-Tyr rich hydrogels which is driven by the material's adhesive property, and additionally confirmed by the suppressed gene expressions of apoptosis and autophagy. On the other hand, HA-Tyr rich hydrogels lead the cells to necrotic death via oxidative stress in quasi-spheroids. This work demonstrates the ideality of HA-Tyr/G-Tyr to modulate cancer cell adhesion, which also has potential in preventing primary metastasis after onco-surgery, biomaterials-based cancer research, and drug testing.

3D printing of inorganic-biopolymer composites for bone regeneration.

In most cases, bone injuries heal without complications, however, there is an increasing number of instances where bone healing needs major clinical intervention. Available treatment options have severe drawbacks, such as donor site morbidity and limited availability for autografting. Bone graft substitutes containing growth factors would be a viable alternative, however they have been associated with dose-related safety concerns and lack control over spatial architecture to anatomically match bone defect sites. A 3D printing offers a solution to produce patient specific bone graft substitutes that are customized to the patient bone defect with temporal control over the incorporated therapeutics to maximize their efficacy. Inspired by the natural constitution of bone tissue, composites made of inorganic phases, such as nanosilicate particles, calcium phosphate, and bioactive glasses, combined with biopolymer matrices have been investigated as building blocks for the biofabrication of bone constructs. Besides capturing elements of the bone physiological structure, these inorganic/organic composites can be designed for specific cohesivity, rheological and mechanical properties, while both inorganic and organic constituents contribute to the composite bioactivity. This review provides an overview of 3D printed composite biomaterial-inks for bone tissue engineering. Furthermore, key aspects in biomaterial-ink design, 3D printing techniques, and the building blocks for composite biomaterial-inks are summarized.

An Antibiotic-Loaded Hydrogel Demonstrates Efficacy as Prophylaxis and Treatment in a Large Animal Model of Orthopaedic Device-Related Infection.

Local antibiotic therapy is increasingly being recognised for its role in preventing and treating orthopaedic device-related infection (ODRI). A bioresorbable, injectable gentamicin-loaded hydrogel has been developed to deliver local antibiotics at the time of surgery with potential for both prevention and treatment of ODRI. In a prophylaxis model, the antibiotic hydrogel was compared with systemic perioperative antibiotic prophylaxis alone in twelve sheep (six per group) at the time of intramedullary (IM) nail insertion to the tibia, which was inoculated with methicillin-sensitive Staphylococcus aureus (MSSA). In a treatment model of single-stage revision surgery, adjunctive antibiotic-loaded hydrogel was compared with systemic antibiotics alone in a single stage revision of MSSA infection associated with a tibia intramedullary nail in eleven sheep (five/six per group). The primary endpoint was quantitative microbiological results of soft tissue, bone and sonicate fluid from explanted hardware at the time of euthanasia. At euthanasia, the control sheep that received no local antibiotics in the prophylaxis model were all culture-positive (median 1x108, range 7x106-3x108 colony forming units, CFU) while only two of six sheep receiving local gentamicin had any culture positive biopsies (median 1x101, range 0 - 1x105 CFU). For the treatment model, sheep receiving only systemic antibiotics were all culture-positive (median 8x105, range 2x103- 9x106 CFU) while only two of six sheep treated with gentamicin-loaded hydrogel had any culture positive biopsies (median 3x102, range 0 - 7x104 CFU). Local gentamicin concentrations measured in extracellular fluid in the tibial canal show a burst release of gentamicin from the hydrogel. Serum gentamicin concentrations peaked in both models at one day post application and were below detection limit thereafter. This study has demonstrated the effective use of a locally delivered antibiotic hydrogel for both the prevention and treatment of ODRI that is superior to that of systemic antibiotics alone. Future studies will endeavour to translate from preclinical to clinical research trials.

Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues.

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. STATEMENT OF SIGNIFICANCE: In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.

Hyaluronic acid-based interpenetrating network hydrogel as a cell carrier for nucleus pulposus repair.

Hyaluronic acid (HA) is a key component of the intervertebral disc (IVD) that is widely investigated as an IVD biomaterial. One persisting challenge is introducing materials capable of supporting cell encapsulation and function, yet with sufficient mechanical stability. In this study, a hybrid interpenetrating polymer network (IPN) was produced as a non-covalent hydrogel, based on a covalently cross-linked HA (HA-BDDE) and HA-poly(N-isopropylacrylamide) (HA-pNIPAM). The hybrid IPN was investigated for its physicochemical properties, with histology and gene expression analysis to determine matrix deposition in vitro and in an ex vivo model. The IPN hydrogel displayed cohesiveness for at least one week and rheological properties resembling native nucleus pulposus (NP) tissue. When implanted in an ex vivo IVD organ culture model, the IPN supported cell viability, phenotype expression of encapsulated NP cells and IVD matrix production over four weeks under physiological loading. Overall, our results indicate the therapeutic potential of this HA-based IPN hydrogel for IVD regeneration.

Bacteriophage Therapy for the Prevention and Treatment of Fracture-Related Infection Caused by Staphylococcus aureus: a Preclinical Study.

Although several studies have shown promising clinical outcomes of phage therapy in patients with orthopedic device-related infections, questions remain regarding the optimal application protocol, systemic effects, and the impact of the immune response. This study provides a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection (FRI) caused by Staphylococcus aureus. In a prevention setting, phage in saline (without any biomaterial-based carrier) was highly effective in the prevention of FRI, compared to systemic antibiotic prophylaxis alone. In the subsequent study involving treatment of established infection, daily administration of phage in saline through a subcutaneous access tube was compared to a single intraoperative application of a phage-loaded hydrogel and a control group receiving antibiotics only. In this setting, although a possible trend of bacterial load reduction on the implant was observed with the phage-loaded hydrogel, no superior effect of phage therapy was found compared to antibiotic treatment alone. The application of phage in saline through a subcutaneous access tube was, however, complicated by superinfection and the development of neutralizing antibodies. The latter was not found in the animals that received the phage-loaded hydrogel, which may indicate that encapsulation of phages into a carrier such as a hydrogel limits their exposure to the adaptive immune system. These studies show phage therapy can be useful in targeting orthopedic device-related infection, however, further research and improvements of these application methods are required for this complex clinical setting. IMPORTANCE Because of the growing spread of antimicrobial resistance, the use of alternative prevention and treatment strategies is gaining interest. Although the therapeutic potential of bacteriophages has been demonstrated in a number of case reports and series over the past decade, many unanswered questions remain regarding the optimal application protocol. Furthermore, a major concern during phage therapy is the induction of phage neutralizing antibodies. This study aimed at providing a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection caused by Staphylococcus aureus. Phage therapy was applied as prophylaxis in a first phase, and as treatment of an established infection in a second phase. The development of phage neutralizing antibodies was evaluated in the treatment study. This study demonstrates that phage therapy can be useful in targeting orthopedic device-related infection, especially as prophylaxis; however, further research and improvements of these application methods are required.

A Hyaluronic Acid Hydrogel Loaded with Gentamicin and Vancomycin Successfully Eradicates Chronic Methicillin-Resistant Staphylococcus aureus Orthopedic Infection in a Sheep Model.

Implantable orthopedic devices have had an enormously positive impact on human health; however, despite best practice, patients are prone to developing orthopedic device-related infections (ODRI) that have high treatment failure rates. One barrier to the development of improved treatment options is the lack of an animal model that may serve as a robust preclinical assessment of efficacy. We present a clinically relevant large animal model of chronic methicillin-resistant Staphylococcus aureus (MRSA) ODRI that persists despite current clinical practice in medical and surgical treatment at rates equivalent to clinical observations. Furthermore, we showed that an injectable, thermoresponsive, hyaluronic acid-based hydrogel loaded with gentamicin and vancomycin outperforms current clinical practice treatment in this model, eliminating bacteria from all animals. These results confirm that local antibiotic delivery with an injectable hydrogel can dramatically increase treatment success rates beyond current clinical practice, with efficacy proven in a robust animal model.

Single-stage revision of MRSA orthopedic device-related infection in sheep with an antibiotic-loaded hydrogel.

Local antimicrobial therapy is an integral aspect of treating orthopedic device-related infection (ODRI), which is conventionally administered via polymethyl-methacrylate (PMMA) bone cement. PMMA, however, is limited by a suboptimal antibiotic release profile and a lack of biodegradability. In this study, we compare the efficacy of PMMA versus an antibiotic-loaded hydrogel in a single-stage revision for chronic methicillin-resistant Staphylococcus aureus (MRSA) ODRI in sheep. Antibiofilm activity of the antibiotic combination (gentamicin and vancomycin) was determined in vitro. Swiss alpine sheep underwent a single-stage revision of a tibial intramedullary nail with MRSA infection. Local gentamicin and vancomycin therapy was delivered via hydrogel or PMMA (n = 5 per group), in conjunction with systemic antibiotic therapy. In vivo observations included: local antibiotic tissue concentration, renal and liver function tests, and quantitative microbiology on tissues and hardware post-mortem. There was a nonsignificant reduction in biofilm with an increasing antibiotic concentration in vitro (p = 0.12), confirming the antibiotic tolerance of the MRSA biofilm. In the in vivo study, four out of five sheep from each treatment group were culture-negative. Antibiotic delivery via hydrogel resulted in 10-100 times greater local concentrations for the first 2-3 days compared with PMMA and were comparable thereafter. Systemic concentrations of gentamicin were minimal or undetectable in both groups, while renal and liver function tests were within normal limits. This study shows that a single-stage revision with hydrogel or PMMA is equally effective, although the hydrogel offers certain practical benefits over PMMA, which make it an attractive proposition for clinical use.

Printability and Shape Fidelity of Bioinks in 3D Bioprinting.

Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance.