Injectable elastin-like recombinamer hydrogels with layered degradation rates for osteochondral regeneration in a rabbit model

Injectable hydrogels have recently emerged as alternatives to sutures for various clinical indications. However, existing injectable hydrogels are unsuitable for hemostasis in minimally invasive surgery because of their weak interfacial adhesion and complex/prolonged processing. Herein, a superwetting injectable hydrogel composed of oppositely charged polysaccharides is developed. The spontaneous spreading of the injectable hydrogel on the surfaces achieves complete wetting and forms tight interfacial contact by absorbing the interfacial water. The superwetting ability and subsequent covalent crosslinking perform fast and ultrastrong wet adhesion (140kPa) on the tissue surface. Ex vivo porcine and in vivo rat models show that the hydrogel successfully leads to the aggregation of erythrocytes for targeted hemostasis (in less than 12 s) without requiring external adjuncts, and no postsurgical adhesions to the peripheral tissues. This further demonstrates that hydrogel can act as an effective hemostasis agent in laparoscopic surgery in a rabbit model. Overall, the strong wet adhesion, antibacterial properties, and easy operability make this injectable hydrogel a promising candidate for hemostasis applications, as it can successfully combine clinical efficacy and transformation opportunities for minimally invasive surgery.

IntroductionThe objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels.MethodChemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques.ResultA 3D scaffold using ELR that combines various peptides with different degradation rates to guide both angiogenesis and neurogenesis has been developed. Notably, scaffolds with different degradation rates promoted distinct patterns of vascularization and innervation, facilitating integration with host tissue. This work demonstrates the potential for tailored tissue engineering, where the scaffold's bioactivities and degradation rates can control angiogenesis and neurogenesis. In an animal model of medication-related osteonecrosis of the jaw (MRONJ), the scaffold showed promising results in promoting bone regeneration in a necrotic environment, as confirmed by histological and imaging analyses. This study opens avenues for novel tissue-engineering strategies where precise control over vascularization and nerve growth is crucial.ConclusionA groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold achieves effective time/space control over necrotic bone regeneration.The authors are grateful for funding from the Spanish Government (PID2020-118669RA-I00)

An in vivo model to study the effect of an injectable hyaluronic acid (HA) hydrogel following puncture-induced lumbar disc injury in rabbits. The aim of this study was to determine the efficacy of an injectable HA hydrogel to maintain disc height and tissue hydration, promote structural repair, and attenuate inflammation and innervation in the lumbar discs. Previously, we have demonstrated that HA hydrogel alleviated inflammation, innervation, and pain to promote disc repair. Nevertheless, the effect of an injectable HA hydrogel in the lumbar disc in a weight-bearing animal model was not performed. We have adopted a surgically puncture-induced disc injury at lumbar levels in a rabbit model. The discs were grouped into sham, puncture with water injection, and puncture with HA hydrogel injection. Postoperatively, we measured changes in disc height using x-ray. We used magnetic resonance imaging to assess disc degeneration on tissue hydration after euthanasia. Post-mortem, we determined histological changes, innervation (PGP9.5) and inflammation (interleukin [IL]-6, IL-1β, and tumor necrosis factor [TNF]-α) in the discs. We have demonstrated a significant reduction of disc height and T2/T1ρ mapping with histological evidence of degenerative discs, increase of innervation and inflammation in puncture-induced disc injury over time. In the HA hydrogel group, disc height was increased at weeks four and eight. A slight increase of T2 mapping, but significantly in T1ρ mapping, was observed in the HA hydrogel group at week 8. We observed homogenous NP distribution and organised AF lamellae at week eight and a slight reduced innervation score in the treatment group. HA hydrogel significantly downregulated IL-6 expression at day 1. This, however, was only slightly reduced for IL-1β and TNF-α. An injectable HA hydrogel had the protective effects in suppressing the loss of disc height, promoting tissue hydration for structural repair, and attenuating inflammation and innervation to prevent further disc degeneration.Level of Evidence: N/A.

A Tribute to Professor Nicholas Peppas.

Repair of osteochondral defects using injectable chitosan-based hydrogel encapsulated synovial fluid-derived mesenchymal stem cells in a rabbit model

Mandibular osteomyelitis (OM) is a challenging disease. Our objective was to assess a new OM model in rabbits induced by arsenic trioxide and to assess the efficacy of local treatment of OM using injectable gentamicin-collagen hydrogels (GNT-COLL). OM was induced unilaterally by controlled confinement of arsenic trioxide paste to the root canal of lower incisors of rabbits, while OM progression was characterized for 16 weeks. On the other hand, two injectable COLL hydrogels functionalized with GNT were prepared and characterized for physicochemical properties; a simple GNT-COLL and a nanohydroxyapatite (nHA)- loaded hydrogel (GNT-COLL/nHA). The two hydrogels were evaluated to treat OM model, while a multidose intramuscular GNT solution served as positive control. Outcomes were assessed by standard methods at 4 and 12 weeks post-surgery. The clinical, radiographical, and histopathological findings provided evidence for the validity of the arsenic-induced OM. The results demonstrated that a single intra-lesional injection of the two hydrogels was more suppressive to OM compared to multidose systemic GNT. The composite GNT-COLL/nHA hydrogel proved to induce early preservation of alveolar bone (ridge) length and higher amount of bone area\total area at 4 weeks (40.53% ± 2.34) followed by GNT-COLL (32.21% ± 0.72). On the other hand, the positive control group revealed the least ridge length and bone area\total area (26.22% ± 1.32) at 4 weeks. Both hydrogels successfully arrested OM with no signs of recurrence for up to 12 weeks. Therefore, results support the greater advantages of the composite hydrogel as an osteogenic/antibiotic delivery system in OM treatment.

Introduction: In-situ photo-cured hydrogels for bone regeneration offer an advantage compared to solid scaffolds or membranes is that it can be used by minimally invasive techniques and can fill irregularly shaped defects easily. Objective: was to prepare an injectable photo-curable hyaluronic acid hydrogel scaffold loaded with bioactive nano-hydroxyapatite using riboflavin as a natural source photoinitiator for bone regeneration and to investigate the effect of addition of nano-hydroxyapatite on the physiochemical and mechanical properties of the prepared hydrogel. Also, the osteogenic potential of the prepared hydrogels was assessed in a rabbit model. Materials and methods: Two groups were prepared, (Group I) photo-cured hyaluronic acid as a control group and (Group II) photo-cured hyaluronic acid/nano-hydroxyapatite. Laboratory characterization tests: FTIR, XRD, SEM, mechanical, swelling and degradation rate tests were performed. Cell viability % using the MTT assay was used to assess the biocompatibility. In vivo bioactivity was assessed in a rabbit model and histomorphometric analysis was done. Results: Statistical analysis of results revealed that the addition of nano-hydroxyapatite increased significantly the mechanical properties of the hydrogels. SEM images demonstrated that the addition of nano-hydroxyapatite caused the formation of inter-connected pores. MTT assay showed that hydrogel extract didn’t affect cell viability after 48h. Histomorphometric analysis results revealed that the photo-cured (GMA-HA/HAP) hydrogel increased the osteogenic potential by one and a half folds compared to the control group and this proved its bioactivity. Conclusion: Results suggest that the prepared photo-cured hyaluronic hydrogel is a promising biomaterial to deliver bioactive nano-hydroxyapatite and has an osteogenic potential.

Uncontrolled bleeding and wound infections following severe trauma pose significant challenges for existing tissue adhesives, primarily due to their weak wet adhesion, slow adhesion formation, cytotoxicity concerns, and lack of antibacterial properties. Herein, an injectable hydrogel (denoted as ES gel) with rapid, robust adhesive sealing and inherent antibacterial activity based on ε-polylysine and a poly(ethylene glycol) derivative is developed. The engineered hydrogel exhibits rapid gelation behavior, high mechanical strength, strong adhesion to various tissues, and can sustain an ultrahigh burst pressure of 450mmHg. It also presents excellent biocompatibility, biodegradability, antibacterial properties, and on-demand removability. Significantly improved hemostatic efficacy of ES gel compared to fibrin glue is demonstrated using various injury models in rats and rabbits. Remarkably, the adhesive hydrogel can effectively halt lethal non-compressible hemorrhages in visceral organs (liver, spleen, and heart) and femoral artery injury models in fully anticoagulated pigs. Furthermore, the hydrogel outperforms commercial products in sutureless wound closure and repair in the rat liver defect, skin incision, and infected full-thickness skin wound models. Overall, this study highlights the promising clinical applications of ES gel for managing uncontrolled hemorrhage, sutureless wound closure, and infected wound repair.

Injectable Elastin-Like Recombinamer Hydrogels with Layered Degradation Rates for Osteochondral Regeneration in a Rabbit Model

Injectable magnetic hydrogel induces multi-programmed cell death and deep tumor regression in magnetic hyperthermia therapy in hepatocellular carcinoma

Injectable bone substitutes and techniques have been developed for use in minimally invasive procedures for bone augmentation. Objective: To develop a novel injectable thermo-sensitive alginate hydrogel (TSAH) as a scaffold to induce bone regeneration, using a minimally invasive tunnelling technique.Material and Methods : An injectable TSAH was prepared from a copolymer solution of 8.0 wt% Poly(N-isopropylacrylamide) (PNIPAAm) and 8.0 wt% AAlg-g-PNIPAAm. In vitro properties of the material, such as its microstructure and the sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2), were investigated. Then, with the subperiosteal tunnelling technique, this material, carrying rhBMP-2, was injected under the labial periosteum of the maxillary anterior alveolar ridge in a rabbit model. New bone formation was evaluated by means of X-ray, micro-computed tomography (micro-CT), fluorescence labelling, histological study, and immunohistochemistry study.Results : The material exhibited good injectability and thermo-irreversible properties. SEM showed an interconnected porous microstructure of the TSAH. The result of ALP activity indicated sustained delivery of BMP-2 from the TSAH from days 3 to 15. In a rabbit model, both TSAH and TSAH/rhBMP-2 induced alveolar ridge augmentation. The percentage of mineralised tissue in the TSAH/rhBMP-2 group (41.6±3.79%) was significantly higher than in the TSAH group (31.3±7.21%; p<0.05). The density of the regenerating tissue was higher in the TSAH/rhBMP-2 group than in the other groups (TSAH group, positive control, blank control; p<0.05).Conclusions : The TSAH provided convenient handling properties for clinical application. To some extent, TSAH could induce ridge augmentation and mineral deposition, which can be enhanced when combined with rhBMP-2 for a minimally invasive tunnelling injection.

Based on our previous study, the utilization of an ultraviolet light photo-cross-linkable hyaluronic acid (HA) hydrogel integrated with a small molecule kartogenin-encapsulated nanoparticles obtained good reconstruction of osteochondral defects in a rabbit model, indicating the superiority of injectable hydrogel-based scaffolds in cartilage tissue engineering. Platelet-rich plasma (PRP), rich in various growth factors, proteins and cytokines, is considered to facilitate cartilage healing by stimulating cell proliferation and inducing chondrogenesis in cartilage defect site. The aim of this study was to test the therapeutic feasibility of autologous PRP combined with injectable HA hydrogel on cartilage repair. The focal cartilage defects with different critical sizes in the medial femoral condyle of a porcine model were used. At 6 months, the minipigs were sacrificed for assessment of macroscopic appearance, magnetic resonance imaging, micro-computed tomography, histology staining and biomechanics. The HA hydrogel combined with PRP-treated group showed more hyaline-like cartilage exhibited by macroscopic appearance and histological staining in terms of extracellular matrix and type II collagen without formation of hypertrophic cartilage, indicating its capacity to improve cartilage healing in the minipig model evaluated at 6 months, with full-thickness cartilage defect of 8.5 mm diameter and osteochondral defect of 6.5 mm diameter, 5 mm depth exhibiting apparent regeneration.

Cartilage regeneration remains a clinical challenge, as existing treatments (including pharmacological and physiological treatments) only alleviate symptoms. This work proposes a novel strategy of in/ex-source synergistic lubrication and develops an injectable bioactive hydrogel that synergistically lubricates articular cartilage and promotes cartilage regenerative repair by providing immediate exogenous biomimetic lubricant and promoting endogenous synovial fluid secretion. The hydrogel, composed of a biomimetic lubricant (HDPA) and platelet-rich-plasma (PRP), rapidly forms a gel via biological cross-linking, offering both physical lubrication and bioremediation functions. It significantly reduces cartilage friction, facilitates cell proliferation, migration or recruitment, and promotes their chondrogenic differentiation. Additionally, it stimulates synovial fibroblasts to secrete endogenous hyaluronic acid, achieving in/ex-source lubrication. RNA-seq reveals suppression of pro-inflammatory NF-κB/IL-1 pathways and activation of TGF-β signaling, promoting hyaluronic acid (HA) synthesis and ECM remodeling. In a rabbit model, the hydrogel induces hyaline cartilage regeneration and achieves the optimal healing effect, outperforming controls and single treatments. The approach of combining a biomimetic lubricant with PRP, therefore, is potentially useful for the treatment of osteoarthritis.

IntroductionIntra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model.MethodsThirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume.ResultsIn terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect.ConclusionRegeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

Injectable hydrogels have attracted increasing attention in tissue regeneration and local drug delivery applications. Current click reactions for preparing injectable hydrogels often require a photoinitiator or catalyst, which may be toxic and may involve complex synthesis of precursors. Here, we report a facile and inexpensive method to prepare injectable and degradable hydrogels via spontaneous amino-yne click reaction without using any initiator or catalyst under physiological conditions based on telechelic electron-deficient dipropiolate ester of polyethylene glycol and water-soluble commercially available carboxymethyl chitosan (CMC). The gelation time, mechanical property, and degradation rate of the hydrogels could be adjusted by varying CMC concentrations and stoichiometric ratios. The reversible pH-induced sol-gel transitions of the hydrogel are presented and the pH-controlled drug release behaviors are demonstrated, of which the mechanism is discussed. In vitro cytotoxicity assays and in vivo in situ injection study of the CMC-based hydrogels showed favorable gel formation, nontoxicity, and good tissue biocompatibility. Therefore, these biodegradable and injectable hydrogels prepared by spontaneous amino-yne click reaction hold potential for tissue engineering and other biomedical applications.