Mechano-Chemical Effect of Gelatin- and HA-Based Hydrogels on Human Retinal Progenitor Cells.

Engineering matrices for cell therapy requires design criteria that include the ability of these materials to support, protect and enhance cellular behavior in vivo. The chemical and mechanical formulation of the biomaterials can influence not only target cell phenotype but also cellular differentiation. In this study, we have demonstrated the effect of a gelatin (Gtn)-hyaluronic acid (HA) hydrogel on human retinal progenitor cells (hRPCs) and show that by altering the mechanical properties of the materials, cellular behavior is altered as well. We have created an interpenetrating network polymer capable of encapsulating hRPCs. By manipulating the stiffness of the hydrogel, the differentiation potential of the hRPCs was controlled. Interpenetrating network 75 (IPN 75; 75% HA) allowed higher expression of rod photoreceptor markers, whereas cone photoreceptor marker expression was found to be higher in IPN 50. In vivo testing of these living matrices performed in Long-Evans rats showed higher levels of rod photoreceptor marker expression when IPN 75 was injected versus IPN 50. These biomaterials mimic biological cues that are required to simulate the dynamic complexity of natural retinal ECM. These hydrogels can be used as a vehicle for cell delivery in vivo as well as for expansion and differentiation in an in vitro 3D system in a highly reproducible manner.

Platelet-Rich Plasma Lysate-Incorporating Gelatin Hydrogel as a Scaffold for Bone Reconstruction.

In implant dentistry, large vertical and horizontal alveolar ridge deficiencies in mandibular and maxillary bone are challenges that clinicians continue to face. One of the limitations of porous blocks for reconstruction of bone in large defects in the oral cavity, and in the musculoskeletal system, is that fibrin clot does not adequately fill the interior pores and does not persist long enough to accommodate cell migration into the center of the block. The objective of our work was to develop a gelatin-based gel incorporating platelet-rich plasma (PRP) lysate, to mimic the role that a blood clot would normally play to attract and accommodate the migration of host osteoprogenitor and endothelial cells into the scaffold, thereby facilitating bone reconstruction. A conjugate of gelatin (Gtn) and hydroxyphenyl propionic acid (HPA), an amino-acid-like molecule, was commended for this application because of its ability to undergo enzyme-mediated covalent cross-linking to form a hydrogel in vivo, after being injected as a liquid. The initiation and propagation of cross-linking were under the control of horseradish peroxidase and hydrogen peroxide, respectively. The objectives of this in vitro study were directed toward evaluating: (1) the migration of rat mesenchymal stem cells (MSCs) into Gtn-HPA gel under the influence of rat PRP lysate or recombinant platelet-derived growth factor (PDGF)-BB incorporated into the gel; (2) the differentiation of MSCs, incorporated into the gel, into osteogenic cells under the influence of PRP lysate and PDGF-BB; and (3) the release kinetics of PDGF-BB from gels incorporating two formulations of PRP lysate and recombinant PDGF-BB. Results: The number of MSCs migrating into the hydrogel was significantly (3-fold) higher in the hydrogel group incorporating PRP lysate compared to the PDGF-BB and the blank gel control groups. For the differentiation/osteogenesis assay, the osteocalcin-positive cell area percentage was significantly higher in both the gel/PRP and gel/PDGF-BB groups, compared to the two control groups: cells in the blank gels grown in cell expansion medium and in osteogenic medium. Results of the ELISA release assay indicated that Gtn-HPA acted as an effective delivery vehicle for the sustained release of PDGF-BB from two different PRP lysate batches, with about 60% of the original PDGF-BB amount in the two groups remaining in the gel at 28 days. Conclusions: Gtn-HPA accommodates MSC migration. PRP-lysate-incorporating hydrogels chemoattract increased MSC migration into the Gtn-HPA compared to the blank gel. PRP-lysate- and the PDGF-BB-incorporating gels stimulate osteogenic differentiation of the MSCs. The release of the growth factors from Gtn-HPA containing PRP lysate can extend over the period of time (weeks) necessary for bone reconstruction. The findings demonstrate that Gtn-HPA can serve as both a scaffold for cell migration and a delivery vehicle that allows sustained and controlled release of the incorporated therapeutic agent over extended periods of time. These findings commend Gtn-HPA incorporating PRP lysate for infusion into porous calcium phosphate blocks for vertical and horizontal ridge reconstruction, and for other musculoskeletal applications.

A bioinspired gelatin-hyaluronic acid-based hybrid interpenetrating network for the enhancement of retinal ganglion cells replacement therapy.

Biomaterial-based cell replacement approaches to regenerative medicine are emerging as promising treatments for a wide array of profound clinical problems. Here we report an interpenetrating polymer network (IPN) composed of gelatin-hydroxyphenyl propionic acid and hyaluronic acid tyramine that is able to enhance intravitreal retinal cell therapy. By tuning our bioinspired hydrogel to mimic the vitreous chemical composition and mechanical characteristics we were able to improve in vitro and in vivo viability of human retinal ganglion cells (hRGC) incorporated into the IPN. In vivo vitreal injections of cell-bearing IPN in rats showed extensive attachment to the inner limiting membrane of the retina, improving with hydrogels stiffness. Engrafted hRGC displayed signs of regenerating processes along the optic nerve. Of note was the decrease in the immune cell response to hRGC delivered in the gel. The findings compel further translation of the gelatin-hyaluronic acid IPN for intravitreal cell therapy.

An Injectable Multifunctional Dual-Phase Bead-Reinforced Gelatin Matrix Permissive of Mesenchymal Stem Cell Infiltration for Musculoskeletal Soft Tissue Repair.

This study develops a novel strategy for regenerative therapy of musculoskeletal soft tissue defects using a dual-phase multifunctional injectable gelatin-hydroxyphenyl propionic acid (Gtn-HPA) composite. The dual-phase gel consists of stiff, degradation-resistant, ≈2-mm diameter spherical beads made from 8 wt% Gtn-HPA in a 2 wt% Gtn-HPA matrix. The results of a 3D migration assay show that both the cell number and migration distance in the dual-phase gel system are comparable with the 2 wt% mono-phase Gtn-HPA, but notably significantly higher than for 8 wt% mono-phase Gtn-HPA (into which few cells migrated). The results also show that the dual phase gel system has degradation resistance and a prolonged growth factor release profile comparable with 8 wt% mono-phase Gtn-HPA. In addition, the compressive modulus of the 2 wt% dual-phase gel system incorporating the 8 wt% bead phase is nearly four-fold higher than the 2 wt% mono-phase gel (5.3 ± 0.4 kPa versus 1.5 ± 0.06 kPa). This novel injectable dual-phase Gtn-HPA composite thus combines the advantages of low-concentration Gtn-HPA (cell migration) with high-concentration Gtn-HPA (stiffness, degradation resistance, slower chemical release kinetics) to facilitate effective reparative/regenerative processes in musculoskeletal soft tissue.

Observation of Collagen-Containing Lesions After Hematoma Resolution in Intracerebral Hemorrhage.

Background and Purpose: The classic presentation of chronic (stage III) hemorrhagic stroke lesions is a fluid-filled cavity. In one of the most commonly used animal models of intracerebral hemorrhage (ICH), we noticed additional solid material within the chronic lesion. We examined the composition of those chronic ICH lesions and compared them with human autopsy cases. Methods: ICH was induced in rats by the injection of collagenase in the striatum. Tissue sections after hematoma resolution corresponding to 3 different chronic time points­28, 42, and 73 to 85 days post-ICH­were selected. Human autopsy reports at the University Hospital of Zurich were searched between 1990 and 2019 for ICH, and 3 chronic cases were found. The rat and human sections were stained with a variety of histopathologic markers. Results: Extensive collagenous material was observed in the chronic lesion after hematoma resolution in both the rat model and human autopsy cases. Additional immunostaining revealed that the material consisted primarily of a loose network of collagen 3 intermingled with occasional GFAP (glial fibrillary acidic protein)-positive processes and collagen 4. Conclusions: A key feature of the chronic ICH lesion is a loose network of collagen 3. The collagenase rat model reproduces the morphology and composition of the chronic human ICH lesion. While identifying new features of ICH lesion pathology, these results are important for treatment and recovery strategies.

Platelet-Derived Growth Factor Stimulated Migration of Bone Marrow Mesenchymal Stem Cells into an Injectable Gelatin-Hydroxyphenyl Propionic Acid Matrix.

Bone marrow mesenchymal stem cells (bMSCs) are responsible in the repair of injured tissue through differentiation into multiple cell types and secretion of paracrine factors, and thus have a broad application profile in tissue engineering/regenerative medicine, especially for the musculoskeletal system. The lesion due to injury or disease may be a closed irregular-shaped cavity deep within tissue necessitating an injectable biomaterial permissive of host (endogenous) cell migration, proliferation and differentiation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) is a natural biopolymer hydrogel which is covalently cross-linked by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in situ and can be delivered to the lesion by needle injection. Growth factors and cytokines can be directly incorporated into the gel or into nano- and micro-particles, which can be employed for sustained release of biomolecules while maintaining their bioactivity. In this study, we selected polyelectrolyte complex nanoparticles (PCNs) prepared with dextran sulfate and chitosan as the carrier for platelet-derived growth factor (PDGF)-BB and stromal cell-derived factor (SDF)-1α, which have been tested effectively in recruiting stem cells. Our in vitro results showed a high degree of viability of bMSCs through the process of Gtn-HPA covalent cross-linking gelation. The Gtn-HPA matrix was highly permissive of bMSC migration, proliferation, and differentiation. PDGF-BB (20 ng/mL) directly incorporated into the gel and, alternatively, released from PCNs stimulated bMSC migration and proliferation. There were only small differences in the results for the direct incorporation of PDGF into the gel compared with its release from PCNs, and for increased doses of the growth factor (200 ng/mL and 2 µg/mL). In contrast, SDF-1α elicited an increase in migration and proliferation only when released from PCNs; its effect on migration was notably less than PDGF-BB. The in vitro results demonstrate that PDGF-BB substantially increases migration of bMSCs into Gtn-HPA and their proliferation in the gel, and that these benefits can be derived from incorporation of a relatively low dose of the growth factor directly into the gel. These findings commend the use of Gtn-HPA/PDGF-BB as an injectable therapeutic agent to treat defects in musculoskeletal tissues.

Controlling Growth Factor Diffusion by Modulating Water Content in Injectable Hydrogels.

Recent advancements in the delivery of therapeutics for retinal diseases include the development of injectable hydrogels, networks of one or more hydrophilic polymers that contain a high-volume fraction of water. These systems are of particular interest due to their biocompatibility, permeability to water-soluble metabolites, and function as minimally invasive injectable delivery vehicles. Recently, hydrogels for ophthalmic applications have been developed that display a controlled release of factors necessary for cellular survival and proliferation. Understanding the relationship between the volume water fraction and the physical, chemical, and diffusion properties of the hydrogel scaffold could aid in the improvement of existing drug delivery treatments for retinal regeneration. In this study, we compared the diffusion and release of human epidermal growth factor (hEGF) encapsulated in different injectable homogenous and heterogenous hydrogels, namely gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and hyaluronic acid-tyramine (HA-Tyr)-based hydrogels. These experimental results were compared with the measured stiffness and water content of these hydrogels and applied to different diffusion theories of polymers to determine the model of best fit. We find that the normalized diffusion and release of hEGF increases with free water content in injectable hydrogels: ranging from 0.176 at 41% free water in HA-Tyr to 0.2 at 53% free water in Gtn-HPA, whereas it decreases with hydrogel stiffness: 600 Pa for Gtn-HPA and 1440 Pa for HA-Tyr. Further, we compared our experimental data with theoretical diffusion models. We found that homogeneous theoretical models, notably the hydrodynamic model (giving a normalized diffusion close to 0.2), provide the most suitable explanation for the measured solute diffusion coefficient. Impact statement Diffusion in a three-dimensional system is a key factor in designing new hydrogel-based materials. It allows to control and predict diffusion in implants and delivery systems. However, very little is done to explore and test the diffusion since it is a complex process. Many models can predict solute diffusion; however, practical application using these models has not yet been done. We have shown the variation of these models in a practical extent, which could have a tremendous impact on designing biomaterial for biological application as it allows one to understand the diffusion of injected drugs and growth factors.

The effects of shock wave stimulation of mesenchymal stem cells on proliferation, migration, and differentiation in an injectable gelatin matrix for osteogenic regeneration.

The treatment of a variety of defects in bony sites could benefit from mitogenic stimulation of osteoprogenitor cells, including endogenous bone marrow-derived mesenchymal stem cells (bMSCs), and from provision of such cells with a matrix permissive of their migration, proliferation, and osteogenic differentiation. That such MSC stimulation could result from treatment with noninvasive (extracorporeal) shock waves (ESWs), and the matrix delivered by injection could enable this therapeutic approach to be employed for applications in which preformed scaffolds and growth factor therapy are difficult to deploy. The objectives of the present study were to investigate focused ESWs for their effects on proliferation, migration, and osteogenic differentiation in an injectable gelatin (Gtn) matrix capable of undergoing covalent cross-linking in vivo. Gtn was conjugated with hydroxyphenyl propionic acid (HPA) in order to enable it to be covalently cross-linked with minute amounts of horseradish peroxidase and hydrogen peroxide. The results demonstrated that 500 shocks of 0.4-mJ/mm2 energy flux density resulted in a twofold greater proliferation of bMSCs in the Gtn-HPA matrix after 14 days, compared with bMSCs grown with supplementation with platelet-derived growth factor (PDGF)-BB, a known mitogen for bMSCs. Moreover, SW treatment enhanced substantially osteogenic differentiation of bMSCs. The Gtn-HPA gel was permissive of MSC migration under the chemotactic influence of the growth factor, PDGF-BB, incorporated into and released by the gel. ESW treatment had no effect on the motility of the MSCs. The findings of the study warrant further investigation of this combined treatment modality for select bony defects.

Extracorporeal Shock Wave Stimulates Angiogenesis and Collagen Production in Facial Soft Tissue.

BACKGROUND: This study investigated the efficacy of extracorporeal shock wave (ESW) application in stimulating dermal thickness, vascularity, and collagen synthesis of facial skin in a large animal model. MATERIALS AND METHODS: The facial skin of the maxillary and mandibular areas of goats (n = 6 per group) was treated with ESWs of different intensities (0.15 and 0.45 mJ/mm2; 1000 pulses). After 4 d, histology and immunohistochemistry were used to evaluate the following: dermal thickness, total number and abundance of microvessels, amount of type 1 collagen, and α-smooth muscle actin expression. RESULTS: Dermal thickness, number and abundance of microvessels, and collagen synthesis increased after ESW application at both intensities (each P < 0.05). When comparing ESW groups, the highest collagen abundance was seen after 0.15 mJ/mm2 (P = 0.034), whereas the highest number of microvessels was detected after treatment with 0.45 mJ/mm2 (P = 0.002). CONCLUSIONS: A single-session application of focused low-energy ESWs to facial skin can increase dermal thickness by stimulating collagen production and local microcirculation. These findings commend the technique for future investigation for pretreatment of local or microvascular skin flaps to enhance tissue healing.

Hydrogel-Based Therapy for Brain Repair After Intracerebral Hemorrhage.

We assessed an injectable gelatin hydrogel containing epidermal growth factor (Gtn-EGF) as a therapy for intracerebral hemorrhage (ICH). ICH was induced in rats via collagenase injection into the striatum. Two weeks later, Gtn-EGF was injected into the cavitary lesion. The hydrogel filled ICH cavities without deforming brain tissue. Immunostaining demonstrated that neural precursor cells could migrate into the matrix, and some of these differentiated into neurons along with the appearance of astrocytes, oligodendrocytes, and endothelial cells. Sensorimotor tests suggested that Gtn-EGF improved neurological recovery. This study provides proof-of-principle that injectable biomaterials may be a translationally relevant approach for treating ICH.

Translational relevance of the goat as a preclinical model of the human labrum and chondrolabral junction-histological study.

The purpose of this study was to evaluate the histologic features of the caprine labrum, with emphasis on the chondrolabral junction, with the goal of informing the feasibility of the goat as an animal model. The left hip joint of six adolescent Spanish goats (Capra pyrenaica) was harvested and subjected to anatomical and histological assessments. Human acetabular and femoral head samples, collected during total hip arthroplasty, served as comparison samples. The caprine labrum was found to consist of mostly type I collagen with uniform crimp, with an average crimp length of 20.8 µm. Upon histological assessment, acetabular articular chondrocytes were found to express substance-P, especially near or in the chondrolabral junction. And the majority of nonvascular cells expressed α-smooth muscle actin (SMA), with no notable elastin and laminin expression. Human labrum demonstrated similar staining patterns. Overall, the goat hip was found to be homologous to the human hip, demonstrating potential as a useful animal model for future studies. This is the first report of a crimped collagen structure in the labrum. Crimped type I collagen at the chondrolabral junction imparts an extension-recovery property which allows for toleration of stress without permanent deformation, underlying the importance of its preservation during surgery. The high expression of substance-P reflects the degree to which the labrum is innervated. Finally, the expression of α-SMA with contractile characteristics could indicate the potential for chondrocyte (i.e., myochondrocytes) modeling of the extracellular matrix. Statement of Clinical Significance: Establishment of a large animal model and deeper knowledge of the histological composition of the hip joint will enhance our study of the acetabular labrum, including repair techniques. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1070-1080, 2020.

Promoting Neuro-Supportive Properties of Astrocytes with Epidermal Growth Factor Hydrogels.

Biomaterials provide novel platforms to deliver stem cell and growth factor therapies for central nervous system (CNS) repair. The majority of these approaches have focused on the promotion of neural progenitor cells and neurogenesis. However, it is now increasingly recognized that glial responses are critical for recovery in the entire neurovascular unit. In this study, we investigated the cellular effects of epidermal growth factor (EGF) containing hydrogels on primary astrocyte cultures. Both EGF alone and EGF-hydrogel equally promoted astrocyte proliferation, but EGF-hydrogels further enhanced astrocyte activation, as evidenced by a significantly elevated Glial fibrillary acidic protein (GFAP) gene expression. Thereafter, conditioned media from astrocytes activated by EGF-hydrogel protected neurons against injury and promoted synaptic plasticity after oxygen-glucose deprivation. Taken together, these findings suggest that EGF-hydrogels can shift astrocytes into neuro-supportive phenotypes. Consistent with this idea, quantitative-polymerase chain reaction (qPCR) demonstrated that EGF-hydrogels shifted astrocytes in part by downregulating potentially negative A1-like genes (Fbln5 and Rt1-S3) and upregulating potentially beneficial A2-like genes (Clcf1, Tgm1, and Ptgs2). Further studies are warranted to explore the idea of using biomaterials to modify astrocyte behavior and thus indirectly augment neuroprotection and neuroplasticity in the context of stem cell and growth factor therapies for the CNS. Stem Cells Translational Medicine 2019;8:1242&1248.

In Situ Cross-linking Hydrogel as a Vehicle for Retinal Progenitor Cell Transplantation.

One of the current limitations of retinal transplantation of stem cells as well as other cell types is the dispersion of cells from the injection site (including loss of cells into the vitreous chamber) and low survival after transplantation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) conjugate is a biodegradable polymer that can undergo covalent cross-linking in situ, allowing for injection of incorporated cells through a small caliber needle followed by gel formation in vivo. We tested the hypothesis that Gtn-HPA hydrogel supports survival and integration of retinal progenitor cells (RPCs) post-transplantation. In vitro compatibility and in vivo graft survival were assessed by mixing an equal volume of Gtn-HPA conjugate and RPC suspension and triggering enzyme-mediated gelation, using minute amounts of horseradish peroxidase and peroxide. Immunocytochemistry showed >80% survival of cells and minimal apoptosis for cells incorporated into Gtn-HPA, equivalent to controls grown on fibronectin-coated flasks. RPCs undergoing mitosis were seen within the three-dimensional Gtn-HPA hydrogel, but the percentage of Ki-67-positive cells was lower compared with the monolayer controls. For in vivo studies, gel-cell mixture or cell suspension in saline was trans-sclerally injected into the left eye of female Long Evans rats immunosuppressed with cyclosporine A. Grafts survived at the 1 week time point of the study, with Gtn-HPA-delivered grafts showing less inflammatory response demonstrated by anti-leukocyte staining. More eyes in the gel-cell mixture group showed surviving cells in the subretinal space compared with saline-delivered controls, while the number of cells surviving per graft was not significantly different between the two groups. This work demonstrates an injectable in situ cross-linking hydrogel as a potential vehicle for stem cell delivery in the retina.

EMSCs Build an All-in-One Niche via Cell-Cell Lipid Raft Assembly for Promoted Neuronal but Suppressed Astroglial Differentiation of Neural Stem Cells.

The therapeutic efficiency of allogenic/intrinsic neural stem cells (NSCs) after spinal cord injury is severely compromised because the hostile niche at the lesion site incurs massive astroglial but not neuronal differentiation of NSCs. Although many attempts are made to reconstruct a permissive niche for nerve regeneration, solely using a living cell material to build an all-in-one, multifunctional, permissive niche for promoting neuronal while inhibiting astroglial differentiation of NSCs is not reported. Here, ectomesenchymal stem cells (EMSCs) are reported to serve as a living, smart material that creates a permissive, all-in-one niche which provides neurotrophic factors, extracellular matrix molecules, cell-cell contact, and favorable substrate stiffness for directing NSC differentiation. Interestingly, in this all-in-one niche, a corresponding all-in-one signal-sensing platform is assembled through recruiting various niche signaling molecules into lipid rafts for promoting neuronal differentiation of NSCs, and meanwhile, inhibiting astrocyte overproliferation through the connexin43/YAP/14-3-3θ pathway. In vivo studies confirm that EMSCs can promote intrinsic NSC neuronal differentiation and domesticating astrocyte behaviors for nerve regeneration. Collectively, this study represents an all-in-one niche created by a single-cell material-EMSCs for directing NSC differentiation.

No effect of topical application of tranexamic acid on articular cartilage.

PURPOSE: The objective of this study was to evaluate potential cytotoxicity of TXA on articular cartilage by assessing chondrocyte viability of osteochondral explants after exposure to different concentrations and durations of TXA. METHODS: Thirty-nine osteochondral plugs (OCPs) were harvested from three adult Yucatan minipigs immediately after their death. OCPs were divided into 13 groups exposed to different concentrations of TXA (1, 2 and 4 mg/ml in saline solution) for 1, 3 and 6 h. Negative controls were exposed to saline solution for 0, 1, 3 and 6 h. Chondrocyte viability was assessed by Live/Dead cell assay and calculated as the ratio of live cells (green fluorescence) to overall cells (green + red cells) for each concentration of TXA and time point in a 50-µm scanned image. RESULTS: No correlation was found between chondrocyte viability, and TXA concentration and time of exposure. Overall, chondrocyte viability ranged from 90 to 99%. There was no statistical difference among control group, 1, 2 and 4 mg/ml TXA solutions at each time point [1 h (n.s.), 3 h (n.s.), 6 h (n.s.)]. Similarly, no statistical difference among groups was observed when comparing cell viability at 1, 3 and 6 h of TXA exposure, (Fig. 2) [1 mg/ml (n.s.), 2 mg/ml (n.s.), and 4 mg/ml (n.s.)]. CONCLUSIONS: In conclusion, doses of TXA approximating the current clinical protocols for topical use did not demonstrate any cytotoxic effects on cartilage explants in a Yucatan mini pig model. Thus, supporting the topical application for procedures with intact cartilage, such as partial knee replacement surgery and cartilage repair procedures.

99mTc-Methyl-Diphosphonate Binding to Mineral Deposits in Cultures of Marrow-Derived Mesenchymal Stem Cells in Osteogenic Medium.

IMPACT STATEMENT: The work is notable for describing a highly sensitive, quantitative, and nondestructive method for evaluating the in vitro amount of mineral accompanying different types of osteogenic differentiation of mesenchymal stem cells in a monolayer cell culture. What is so unique and useful about the method is that it has the potential to be used to define the kinetics of the differentiation process, reflected in the mineralization, without destroying the monolayer. Therefore, it remains intact for further experiments.