Assessing Bioprinted Functionalized Grafts for Biological Tendon Augmentation In Vitro.

Tendinopathy, characterized by inflammatory and degenerative changes, presents challenges in sports and medicine. In addressing the limitations of conservative management, this study focuses on developing tendon grafts using extrusion bioprinting with platelet-rich plasma (PRP)-infused hydrogels loaded with tendon cells. The objective is to understand paracrine interactions initiated by bioprinted tendon grafts in either inflamed or non-inflamed host tissues. PRP was utilized to functionalize methacrylate gelatin (GelMA), incorporating tendon cells for graft bioprinting. Bioinformatic analyses of overexpressed proteins, predictive of functional enrichment, revealed insights into PRP graft behavior in both non-inflamed and inflamed environments. PRP grafts activated inflammatory pathways, including Interleukin 17 (IL-17), neuroinflammation, Interleukin 33 (IL-33), and chemokine signaling. Interleukin 1 beta (IL-1b) in the graft environment triggered p38 mitogen-activated protein kinase (MAPK) signaling, nuclear factor kappa light chain enhancer of activated B cells (NF-kB) canonical pathway, and Vascular Endothelial Growth Factor (VEGF) signaling. Biological enrichment attributed to PRP grafts included cell chemotaxis, collagen turnover, cell migration, and angiogenesis. Acellular PRP grafts differed from nude grafts in promoting vessel length, vessel area, and junction density. Angiogenesis in cellular grafts was enhanced with newly synthesized Interleukin 8 (IL-8) in cooperation with IL-1b. In conclusion, paracrine signaling from PRP grafts, mediated by chemokine activities, influences cell migration, inflammation, and angiogenic status in host tissues. Under inflammatory conditions, newly synthesized IL-8 regulates vascularization in collaboration with PRP.

Activity recovery for differently shaped objects in quantitative SPECT.

Objective.The aim was to theoretically and experimentally investigate recovery in SPECT images with objects of different shapes. Furthermore, the accuracy of volume estimation by thresholding was studied for those shapes.Approach.Nine spheres, nine oblate spheroids, and nine prolate spheroids phantom inserts were used, of which the six smaller spheres were part of the NEMA IEC body phantom and the rest of the inserts were 3D-printed. The inserts were filled with99mTc and177Lu. When filled with99mTc, SPECT images were acquired in a Siemens Symbia Intevo Bold gamma camera and when filled with177Lu in a General Electric NM/CT 870 DR gamma camera. The signal rate per activity (SRPA) was determined for all inserts and represented as a function of the volume-to-surface ratio and of the volume-equivalent radius using VOIs defined according to the sphere dimensions and VOIs defined using thresholding. Experimental values were compared with theoretical curves obtained analytically (spheres) or numerically (spheroids), starting from the convolution of a source distribution with a point-spread function. Validation of the activity estimation strategy was performed using four 3D-printed ellipsoids. Lastly, the threshold values necessary to determine the volume of each insert were obtained.Main results.Results showed that SRPA values for the oblate spheroids diverted from the other inserts, when SRPA were represented as a function of the volume-equivalent radius. However, SRPA values for all inserts followed a similar behaviour when represented as a function of the volume-to-surface ratio. Results for ellipsoids were in agreement with those results. For the three types of inserts the volume could be accurately estimated using a threshold method for volumes larger than 25 ml.Significance.Determination of SRPA independently of lesion or organ shape should decrease uncertainties in estimated activities and thereby, in the long term, be beneficial to patient care.

Implementation of an In-House 3D Manufacturing Unit in a Public Hospital's Radiology Department.

OBJECTIVE: Three-dimensional printing has become a leading manufacturing technique in healthcare in recent years. Doubts in published studies regarding the methodological rigor and cost-effectiveness and stricter regulations have stopped the transfer of this technology in many healthcare organizations. The aim of this study was the evaluation and implementation of a 3D printing technology service in a radiology department. METHODS: This work describes a methodology to implement a 3D printing service in a radiology department of a Spanish public hospital, considering leadership, training, workflow, clinical integration, quality processes and usability. RESULTS: The results correspond to a 6-year period, during which we performed up to 352 cases, requested by 85 different clinicians. The training, quality control and processes required for the scaled implementation of an in-house 3D printing service are also reported. CONCLUSIONS: Despite the maturity of the technology and its impact on the clinic, it is necessary to establish new workflows to correctly implement them into the strategy of the health organization, adjusting it to the needs of clinicians and to their specific resources. SIGNIFICANCE: This work allows hospitals to bridge the gap between research and 3D printing, setting up its transfer to clinical practice and using implementation methodology for decision support.

Wound-Microenvironment Engineering through Advanced-Dressing Bioprinting.

In patients with comorbidities, a large number of wounds become chronic, representing an overwhelming economic burden for healthcare systems. Engineering the microenvironment is a paramount trend to activate cells and burst-healing mechanisms. The extrusion bioprinting of advanced dressings was performed with novel composite bioinks made by blending adipose decellularized extracellular matrix with plasma and human dermal fibroblasts. Rheological and microstructural assessments of the composite hydrogels supported post-printing cell viability and proliferation over time. Embedded fibroblasts expressed steady concentrations of extracellular matrix proteins, including type 1, 3 and 4 collagens and fibronectin. ELISA assessments, multiplex protein arrays and ensuing bioinformatic analyses revealed paracrine activities corresponding to wound-healing activation through the modulation of inflammation and angiogenesis. The two modalities of advanced dressings, differing in platelet number, showed differences in the release of inflammatory and angiogenic cytokines, including interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conditioned media stimulated human-dermal-cell proliferation over time. Our findings open the door to engineering the microenvironment as a strategy to enhance healing.

Unraveling the Signaling Secretome of Platelet-Rich Plasma: Towards a Better Understanding of Its Therapeutic Potential in Knee Osteoarthritis.

Platelets and their secretory products play an important role in determining the balance between tissue repair and tissue damage. To obtain novel insights into the molecular composition of platelet-rich plasma (PRP) and contextualize them in knee osteoarthritis (OA), two different plasma formulations, namely PRP and platelet-poor plasma (PPP), were prepared from six healthy donors following a biobank-automated protocol. Inter-donor differences were analyzed, and pools were created before performing multiplexing protein arrays. In addition, PRP and PPP were prepared from six patients following our in-house protocols. Supernatants from PRP and PPP were harvested one hour after calcium chloride activation. Multiplexing protein arrays were performed in parallel for all plasma formulations. Results were normalized to fold change in relation to PPP and examined using Ingenuity Pathway Analysis Software. Bioinformatic predictions showed that PRPs constitute a signaling system with interrelated networks of inflammatory and angiogenic proteins, including but not limited to interleukin-6 and -8 (IL-6, IL-8), insulin like growth factor 1 (IGF-1), transforming growth factor beta, (TGF-b), and vascular endothelial growth factor (VEGF) signaling, underlying biological actions. Predictions of canonical systems activated with PRP molecules include various inflammatory pathways, including high-mobility group box protein (HMGB1) and interleukin 17 (IL-17) signaling, neuroinflammation, and nuclear factor-kappa b (NF-κB) pathways. Eventually, according to these predictions and OA evolving knowledge, selected PRP formulations should be tailored to modulate different inflammatory phenotypes, i.e., meta-inflammation, inflame-aging or posttraumatic inflammatory osteoarthritis. However, further research to discriminate the peculiarities of autologous versus allogeneic formulations and their effects on the various OA inflammatory phenotypes is needed to foster PRPs.

Plasma-Based Bioinks for Extrusion Bioprinting of Advanced Dressings.

Extrusion bioprinting based on the development of novel bioinks offers the possibility of manufacturing clinically useful tools for wound management. In this study, we show the rheological properties and printability outcomes of two advanced dressings based on platelet-rich plasma (PRP) and platelet-poor plasma (PPP) blended with alginate and loaded with dermal fibroblasts. Measurements taken at 1 h, 4 days, and 18 days showed that both the PRP- and PPP-based dressings retain plasma and platelet proteins, which led to the upregulation of angiogenic and immunomodulatory proteins by embedded fibroblasts (e.g., an up to 69-fold increase in vascular endothelial growth factor (VEGF), an up to 188-fold increase in monocyte chemotactic protein 1 (MCP-1), and an up to 456-fold increase in hepatocyte growth factor (HGF) 18 days after printing). Conditioned media harvested from both PRP and PPP constructs stimulated the proliferation of human umbilical vein endothelial cells (HUVECs), whereas only those from PRP dressings stimulated HUVEC migration, which correlated with the VEGF/MCP-1 and VEGF/HGF ratios. Similarly, the advanced dressings increased the level of interleukin-8 and led to a four-fold change in the level of extracellular matrix protein 1. These findings suggest that careful selection of plasma formulations to fabricate wound dressings can enable regulation of the molecular composition of the microenvironment, as well as paracrine interactions, thereby improving the clinical potential of dressings and providing the possibility to tailor each composition to specific wound types and healing stages.

Overview of Current Advances in Extrusion Bioprinting for Skin Applications.

Bioprinting technologies, which have the ability to combine various human cell phenotypes, signaling proteins, extracellular matrix components, and other scaffold-like biomaterials, are currently being exploited for the fabrication of human skin in regenerative medicine. We performed a systematic review to appraise the latest advances in 3D bioprinting for skin applications, describing the main cell phenotypes, signaling proteins, and bioinks used in extrusion platforms. To understand the current limitations of this technology for skin bioprinting, we briefly address the relevant aspects of skin biology. This field is in the early stage of development, and reported research on extrusion bioprinting for skin applications has shown moderate progress. We have identified two major trends. First, the biomimetic approach uses cell-laden natural polymers, including fibrinogen, decellularized extracellular matrix, and collagen. Second, the material engineering line of research, which is focused on the optimization of printable biomaterials that expedite the manufacturing process, mainly involves chemically functionalized polymers and reinforcement strategies through molecular blending and postprinting interventions, i.e., ionic, covalent, or light entanglement, to enhance the mechanical properties of the construct and facilitate layer-by-layer deposition. Skin constructs manufactured using the biomimetic approach have reached a higher level of complexity in biological terms, including up to five different cell phenotypes and mirroring the epidermis, dermis and hypodermis. The confluence of the two perspectives, representing interdisciplinary inputs, is required for further advancement toward the future translation of extrusion bioprinting and to meet the urgent clinical demand for skin equivalents.

Freeze-Drying of Platelet-Rich Plasma: The Quest for Standardization.

The complex biology of platelets and their involvement in tissue repair and inflammation have inspired the development of platelet-rich plasma (PRP) therapies for a broad array of medical needs. However, clinical advances are hampered by the fact that PRP products, doses and treatment protocols are far from being standardized. Freeze-drying PRP (FD-PRP) preserves platelet function, cytokine concentration and functionality, and has been proposed as a consistent method for product standardization and fabrication of an off-the-shelf product with improved stability and readiness for future uses. Here, we present the current state of experimental and clinical FD-PRP research in the different medical areas in which PRP has potential to meet prevailing medical needs. A systematic search, according to PRISMA (Preferred Reported Items for Systematic Reviews and Meta-Analyses) guidelines, showed that research is mostly focused on wound healing, i.e., developing combination products for ulcer management. Injectable hydrogels are investigated for lumbar fusion and knee conditions. In dentistry, combination products permit slow kinetics of growth factor release and functionalized membranes for guided bone regeneration.

Wound Dressing Selection Is Critical to Enhance Platelet-Rich Fibrin Activities in Wound Care.

The use of platelet-rich fibrin (PRF) is investigated in ulcer management because it provides a healing milieu rich in growth factors and cytokines. Although crucial, the relevance of secondary dressings is under-researched and no data support the use of any particular dressing in preference to another. We assessed the properties of different dressing categories, including alginates, hydrocolloids, foams, hydrofibers, films, meshes and gauzes, in terms of affinity for PRF, releasate management (retention/extrusion) and the kinetics of cytokine release as well as the influence of each combination product, [PRF + dressing], on dermal cell behaviour, aiming to provide useful information for choosing the most adequate dressing for each particular patient. Active dressings including alginates, hydrofibers, foams and hydrocolloids blend with PRF, creating a diverse combination of products with different performances. Alginate and hydrofiber showed the highest affinity but moderate retention of releasate, without interfering with cell functions. Instead, the foam sequestered the releasate and hindered the release of growth factors, thereby compromising cell activities. Film and mesh presented very poor releasate retention and performed similarly to PRF by itself. Affinity index and releasate management explained 79% of platelet-derived growth factor (PDGF-BB) concentration variability, p < 0.001. Cell proliferation depended on the ability of the combination product to retain/release supernatant, PDGF-BB concentration and cell adhesion R2 = 0.91, p = 0.014.

Image-based Characterization of 3D Collagen Networks and the Effect of Embedded Cells.

Collagen microstructure is closely related to the mechanical properties of tissues and affects cell migration through the extracellular matrix. To study these structures, three-dimensional (3D) in vitro collagen-based gels are often used, attempting to mimic the natural environment of cells. Some key parameters of the microstructure of these gels are fiber orientation, fiber length, or pore size, which define the mechanical properties of the network and therefore condition cell behavior. In the present study, an automated tool to reconstruct 3D collagen networks is used to extract the aforementioned parameters of gels of different collagen concentration and determine how their microstructure is affected by the presence of cells. Two different experiments are presented to test the functionality of the method: first, collagen gels are embedded within a microfluidic device and collagen fibers are imaged by using confocal fluorescence microscopy; second, collagen gels are directly polymerized in a cell culture dish and collagen fibers are imaged by confocal reflection microscopy. Finally, we investigate and compare the collagen microstructure far from and in the vicinities of MDA-MB 23 cells, finding that cell activity during migration was able to strongly modify the orientation of the collagen fibers and the porosity-related values.

Matrix architecture plays a pivotal role in 3D osteoblast migration: The effect of interstitial fluid flow.

Osteoblast migration is a crucial process in bone regeneration, which is strongly regulated by interstitial fluid flow. However, the exact role that such flow exerts on osteoblast migration is still unclear. To deepen the understanding of this phenomenon, we cultured human osteoblasts on 3D microfluidic devices under different fluid flow regimes. Our results show that a slow fluid flow rate by itself is not able to alter the 3D migratory patterns of osteoblasts in collagen-based gels but that at higher fluid flow rates (increased flow velocity) may indirectly influence cell movement by altering the collagen microstructure. In fact, we observed that high fluid flow rates (1 µl/min) are able to alter the collagen matrix architecture and to indirectly modulate the migration pattern. However, when these collagen scaffolds were crosslinked with a chemical crosslinker, specifically, transglutaminase II, we did not find significant alterations in the scaffold architecture or in osteoblast movement. Therefore, our data suggest that high interstitial fluid flow rates can regulate osteoblast migration by means of modifying the orientation of collagen fibers. Together, these results highlight the crucial role of the matrix architecture in 3D osteoblast migration. In addition, we show that interstitial fluid flow in conjunction with the matrix architecture regulates the osteoblast morphology in 3D.

Quantification of angiogenic sprouting under different growth factors in a microfluidic platform.

Angiogenesis, as example of collective migration of endothelial cells (ECs), is the main dynamic process that culminates in sprout formation from existing vessels. After tissue injury, the vascularity is interrupted, triggering the regeneration process and the release of different growth factors (GFs). The main aim of this work is to quantify the effect of specific GFs during the initial stage of sprout formation, namely: VEGF, PDGF-BB, TGFß and BMP-2, all of them involved in regenerative processes. For this purpose, we designed a novel algorithm implemented in Matlab to quantify the advance of the EC monolayer over time and the sprout structure in 3D. Our results show that VEGF is the main regulatory GF on angiogenesis processes, producing longer sprouts with higher frequency. However, the chemoattractant effect of VEGF depends on its concentration and its spatiotemporal location, having a critical impact on the sprout time evolution. PDGF-BB (namely as PDGF) has a global negative effect on both the number and length of sprouts. TGFß enhances sprout length at earlier times, although its effect gradually disappears over time. Finally, BMP-2 produces, overall, less number and shorter sprouts, but was the only GF with a positive evolution at longer times, producing fewer but longer sprouts.

Characterization of Fibrin and Collagen Gels for Engineering Wound Healing Models.

Hydrogels are used for 3D in vitro assays and tissue engineering and regeneration purposes. For a thorough interpretation of this technology, an integral biomechanical characterization of the materials is required. In this work, we characterize the mechanical and functional behavior of two specific hydrogels that play critical roles in wound healing, collagen and fibrin. A coherent and complementary characterization was performed using a generalized and standard composition of each hydrogel and a combination of techniques. Microstructural analysis was performed by scanning electron microscopy and confocal reflection imaging. Permeability was measured using a microfluidic-based experimental set-up, and mechanical responses were analyzed by rheology. We measured a pore size of 2.84 and 1.69 µm for collagen and fibrin, respectively. Correspondingly, the permeability of the gels was 1.00·10-12 and 5.73·10-13 m2. The shear modulus in the linear viscoelastic regime was 15 Pa for collagen and 300 Pa for fibrin. The gels exhibited strain-hardening behavior at ca. 10% and 50% strain for fibrin and collagen, respectively. This consistent biomechanical characterization provides a detailed and robust starting point for different 3D in vitro bioapplications, such as collagen and/or fibrin gels. These features may have major implications for 3D cellular behavior by inducing divergent microenvironmental cues.