Pello Sánchez

Encapsulation of Cells in Alginate Gels

Cell microencapsulation is based on the immobilization of cells for continuous release of therapeutics. This approach has been tested in the treatment of many diseases and several clinical trials have been performed. Factors such as the choice of cells to be encapsulated, the biomaterial used, and the procedure for carrying out the capsules are important issues when implementing this technology.This book chapter makes a comprehensive description of alginate, the most frequently employed biomaterial, passing by its structure, the extraction and treatment, and finishing with the process of gelation. It also describes the various modifications that can be carried out to allow the interaction between the alginate and the integrin receptors of encapsulated cells. The main microencapsulation technologies are presented as well as how 100 μm alginate-Poly-L-Lysine-alginate microcapsules can be fabricated with Flow-focusing technology.

Ultrasound-guided plasma rich in growth factors injections and scaffolds hasten motor nerve functional recovery in an ovine model of nerve crush injury.

In the present study we evaluated the motor recovery process of peripheral nerve injury (PNI), based on electrophysiological and histomorphometric criteria, after treatment with plasma rich in growth factors (PRGF) injections and scaffolds in an ovine model. Three groups of sheep underwent a nerve crush lesion: the first group (n = 3) was left to recover spontaneously (SR); the second group was administered saline injections (SI; n = 5) and a third group (n = 6) received PRGF injections and scaffolds immediately after the crush injury. At post‐intervention week 8, 70% of sheep in the PRGF group were CMAP‐positive, with no electrophysiological response in the rest of the groups. Histomorphometric analysis 12 weeks after the surgical intervention revealed that the average axonal density of the SR (1184 ± 864 axons/µm2) and SI (3109 ± 2450 axons/µm2) groups was significantly inferior to the control (8427 ± 2433 axons/µm2) and also inferior to the PRGF group (5276 ± 4148 axons/µm2), showing no significant differences between the control and PRGF groups. The axonal size of the SR and SI groups was significantly smaller compared with the control group (18 ± 4 µm2), whereas the axonal size of the PRGF group (6 ± 5 µm2) did not show statistical differences from the control. Morphometry of the target muscles indicated that the PRGF group had the lowest percentage volume reduction 12 weeks after the crush injury. The PRGF group had larger muscle fibre areas than the SI and SR groups, although the differences did not reach statistical significance. Overall, these data suggest that the PRGF injections and scaffolds hastened functional axon recovery and dampened atrophy of the target muscles in an ovine model. Copyright

Muscle repair: platelet-rich plasma derivates as a bridge from spontaneity to intervention.

Muscle injuries account for between 10% and 55% of all sporting injuries. Although the skeletal muscle is a plastic organ capable of responding efficiently to environmental changes, the appropriate treatment of muscle injuries remains a daunting clinical challenge in sports medicine. There is considerable evidence to indicate that growth factors, such as transforming growth factor-β (TGFβ), hepatocyte growth factor (HGF) or insulin-like growth factor (IGF), and fibrin matrix are key in cellular events required for muscle repair and regeneration, namely myogenesis, angiogenesis and fibrogenesis. An innovative biological approach to the treatment of muscle injuries is the application of Plasma Rich in Growth Factors (PRGF) in intramuscular infiltrations. PRGF delivers growth factors, cytokines and adhesive proteins present in platelets and plasma, as well as other biologically-active proteins conveyed by the plasma, such as fibrinogen, prothrombin and fibronectin. This autologous, mimetic biomaterial embedded with a pool of growth factors acts as a smart dynamic scaffold, and should be applied taking into account a biological approach. A clinical trial is required to assess the functional repair outcome of PRGF infiltrations in muscle injuries.

The Properties of 3 Different Plasma Formulations and Their Effects on Tendinopathic Cells

Background: Tendinopathies are attributed to failure of the healing process and inadequate tissue remodeling. Plasma injections can trigger regenerative responses by modifying the molecular microenvironment. Purpose: To examine the differences in the mitotic, chemotactic, anabolic, and inflammatory effects between leukocyte- and platelet-rich plasma (L-PRP), platelet-rich plasma (PRP), and platelet-poor plasma (PPP). Study Design: Controlled laboratory study. Methods: Tendinopathic cells were cultured in 3-dimensional (3D) hydrogels formed using PPP, PRP, and L-PRP. Cell migration was evaluated using a μ-Slide chemotaxis chamber with video microscopy. Proliferation was assessed using XTT assays. Expression of genes associated with matrix turnover, including type 1 collagen (COL1A1), COL3A1, aggrecan, decorin, fibronectin, matrix metalloproteinase 1 (MMP-1), MMP-3, A Disintegrin-Like And Metalloprotease With Thrombospondin Type 1 Motif proteins 4 (ADAMTS-4), and ADAMTS-5, was assessed using real-time reverse-transcription polymerase chain reaction. Secreted inflammatory proteins, including interleukin (IL)–1β, IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES), as well as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF), were quantified using enzyme-linked immunosorbent assay. Results: Tendinopathic cells migrate at a higher velocity along L-PRP and PRP than along PPP gradients. PRP and L-PRP promote hypercellularity. PPP and PRP showed more pronounced anabolic properties, as demonstrated by enhanced COL1A1 and COL3A1 and reduced MMP-1 expression. Decorin, fibronectin, and aggrecan were downregulated in L-PRP compared with PPP and PRP. L-PRP and PRP were shown to be more proinflammatory than PPP in terms of IL-6 secretion, but cells in PPP showed MCP-1high phenotype. CTGF secretion was significantly reduced in L-PRP compared with PPP and PRP. Conclusion: The main advantages of L-PRP and PRP use, compared with PPP, include their stronger chemotactic and proliferative properties. While PPP and PRP stimulate matrix anabolism, L-PRP is more proinflammatory. Emphasis should be placed on the temporal needs and biological characteristics of injured tendons, and plasma formulations need to be tailored accordingly. Clinical Relevance: Versatile systems allowing the preparation of different plasma formulations, such as PPP, PRP, or L-PRP, can help refine clinical applications by taking advantage of their different biological properties.

Platelet rich plasma and knee surgery.

In orthopaedic surgery and sports medicine, the knee joint has traditionally been considered the workhorse. The reconstruction of every damaged element in this joint is crucial in achieving the surgeons goal to restore the knee function and prevent degeneration towards osteoarthritis. In the last fifteen years, the field of regenerative medicine is witnessing a boost of autologous blood-derived platelet rich plasma products (PRPs) application to effectively mimic and accelerate the tissue healing process. The scientific rationale behind PRPs is the delivery of growth factors, cytokines, and adhesive proteins present in platelets and plasma, as well as other biologically active proteins conveyed by the plasma such as fibrinogen, prothrombin, and fibronectin; with this biological engineering approach, new perspectives in knee surgery were opened. This work describes the use of PRP to construct and repair every single anatomical structure involved in knee surgery, detailing the process conducted in ligament, meniscal, and chondral surgery.

Intraosseous Infiltration of Platelet-Rich Plasma for Severe Knee Osteoarthritis

We describe a new technique of platelet-rich plasma (PRP) infiltration for the treatment of severe knee osteoarthritis. PRP intra-articular infiltration is a promising treatment for knee osteoarthritis, but it still has some limitations in high-degree osteoarthritis. Diagnosis of osteoarthritis is based on clinical and radiographic findings, and patients with grade III or IV knee tibiofemoral osteoarthritis based on the Ahlbäck scale are considered candidates for this technique. The technique consists of performing intraosseous infiltration of PRP into the subchondral bone, which acts on this tissue and consequently on cartilage-bone communication. Although the intraosseous injection hinders the conventional knee intra-articular infiltration, it allows an extension of the range of action of the PRP, which acts directly on the subchondral bone, which is involved in the progression of osteoarthritis. Thus this technique involves a new administration of PRP that can delay knee arthroplasty; moreover, it can be applied for not only severe osteoarthritis but also other pathologies in which the subchondral bone is critical in the etiology, such as necrosis and osteochondral lesions.

Three-Dimensional Platelet-Rich Plasma Hydrogel Model to Study Early Tendon Healing

Since the experimental conditions of cell cultures may bias results, it is critical to use suitable models. This is also true in the context of tendon cell biology and the study of platelet-rich plasma (PRP) therapies and PRP-augmented cell-based therapies. We compared the culture of human tendon cells in 2 dimensions (2D) with PRP-supplemented media to culture in matching 3-dimensional (3D) PRP hydrogels. Cell proliferation, cell shape, and the pattern of gene and protein expression were examined. Our data revealed modifications in cell shape and enhanced expression of tenomodulin and scleraxis in 3D hydrogels. Additionally, protein secretion analysis using glass-based arrays specific for angiogenesis revealed differences in interleukin (IL)-6 and IL-8 protein expression between 2D cultures and 3D hydrogels, while the secretion of other angiogenic or inflammatory cytokines was unaffected. Our study suggests that 3D hydrogels are physiologically more relevant than 2D cultures in the study of tendon cells, based on cell shape, support of tenocyte proliferation, phenotype, and the pattern of gene and protein expression.

Combination of Intra-Articular and Intraosseous Injections of Platelet Rich Plasma for Severe Knee Osteoarthritis: A Pilot Study

The aim of this study was to assess a novel approach to treating severe knee osteoarthritis by targeting synovial membrane, superficial articular cartilage, synovial fluid, and subchondral bone by combining intra-articular injections and intraosseous infiltrations of platelet rich plasma. We explored a new strategy consisting of intraosseous infiltrations of platelet rich plasma into the subchondral bone in combination with the conventional intra-articular injection in order to tackle several knee joint tissues simultaneously. We assessed the clinical outcomes through osteoarthritis outcome score (KOOS) and the inflammatory response by quantifying mesenchymal stem cells in synovial fluid. There was a significant pain reduction in the KOOS from baseline (61.55 ± 14.11) to week 24 (74.60 ± 19.19), after treatment (p = 0.008), in the secondary outcomes (symptoms, p = 0.004; ADL, p = 0.022; sport/rec., p = 0.017; QOL, p = 0.012), as well as VAS score (p < 0.001) and Lequesne Index (p = 0.008). The presence of mesenchymal stem cells in synovial fluid and colony-forming cells one week after treatment decreased substantially from 7.98 ± 8.21 MSC/μL to 4.04 ± 5.36 MSC/μL (p = 0.019) and from 601.75 ± 312.30 to 139.19 ± 123.61  (p = 0.012), respectively. Intra-articular injections combined with intraosseous infiltrations of platelet rich plasma reduce pain and mesenchymal stem cells in synovial fluid, besides significantly improving knee joint function in patients with severe knee osteoarthritis. This trial is registered on EudraCT with the number 2013-003982-32.

Modulation of Synovial Fluid-Derived Mesenchymal Stem Cells by Intra-Articular and Intraosseous Platelet Rich Plasma Administration

The aim of this study was to evaluate the effect of intra-articular (IA) or a combination of intra-articular and intraosseous (IO) infiltration of Platelet Rich Plasma (PRP) on the cellular content of synovial fluid (SF) of osteoarthritic patients. Thirty-one patients received a single infiltration of PRP either in the IA space (n = 14) or in the IA space together with two IO infiltrations, one in the medial femoral condyle and one in the tibial plateau (n = 17). SF was collected before and after one week of the infiltration. The presence in the SF of mesenchymal stem cells (MSCs), monocytes, and lymphocytes was determined and quantified by flow cytometry. The number and identity of the MSCs were further confirmed by colony-forming and differentiation assays. PRP infiltration into the subchondral bone (SB) and the IA space induced a reduction in the population of MSCs in the SF. This reduction in MSCs was further confirmed by colony-forming (CFU-F) assay. On the contrary, IA infiltration alone did not cause variations in any of the cellular populations by flow cytometry or CFU-F assay. The SF of osteoarthritic patients contains a population of MSCs that can be modulated by PRP infiltration of the SB compartment.

Platelet Rich Plasma (PRP) Biotechnology: Concepts and Therapeutic Applications in Orthopedics and Sports Medicine

Regenerative medicine is the augmentation or substitution of diseased or injured cells or tissues by one of two means: (1) an improvement in the ability of endogenous cells to reform damaged tissue or (2) the use of exogenous cells or tissues to replace damaged cells or tissues. Advances in regenerative medicine essentially depend on improving our understanding of cell biology and molecular signaling. Cell signaling is complex and incompletely understood due to the multiple interactions and cross-talk among system components. The human body has some 100 trillion cells, which in the healthy state coordinate their actions through an exchange of chemical signals to maintain body homeostasis. Every cell phenotype secretes signaling proteins that influence their own behavior (autocrine) or the behavior of other neighboring cells (paracrine) through interactions with specific transmembrane receptors located in the cellular membrane. Currently, a great deal of research is directed towards improving our understanding of intercellular communication and the intracellular transduction of these signals; in the field of regenerative medicine, this knowledge will help to disentangle the mysteries of tissue repair and to achieve proper tissue repair and regeneration. Moreover, to reach this goal we must integrate all the information and understanding derived from basic research into novel therapies that yield quicker and more efficient tissue regeneration.