Transit Amplifying Cells (TACs): a still not fully understood cell population.

Maintenance of tissue homeostasis and tissue regeneration after an insult are essential functions of adult stem cells (SCs). In adult tissues, SCs proliferate at a very slow rate within "stem cell niches", but, during tissue development and regeneration, before giving rise to differentiated cells, they give rise to multipotent and highly proliferative cells, known as transit-amplifying cells (TACs). Although differences exist in diverse tissues, TACs are not only a transitory phase from SCs to post-mitotic cells, but they also actively control proliferation and number of their ancestor SCs and proliferation and differentiation of their progeny toward tissue specific functional cells. Autocrine signals and negative and positive feedback and feedforward paracrine signals play a major role in these controls. In the present review we will consider the generation and the role played by TACs during development and regeneration of lining epithelia characterized by a high turnover including epidermis and hair follicles, ocular epithelial surfaces, and intestinal mucosa. A comparison between these different tissues will be made. There are some genes and molecular pathways whose expression and activation are common to most TACs regardless their tissue of origin. These include, among others, Wnt, Notch, Hedgehog and BMP pathways. However, the response to these molecular signals can vary in TACs of different tissues. Secondly, we will consider cultured cells derived from tissues of mesodermal origin and widely adopted for cell therapy treatments. These include mesenchymal stem cells and dedifferentiated chondrocytes. The possible correlation between cell dedifferentiation and reversion to a transit amplifying cell stage will be discussed.

Trojan-horse silk fibroin nanocarriers loaded with a re-call antigen to redirect immunity against cancer.

BACKGROUND: The current challenge for immunotherapies is to generate effective antitumor immunity. Since tumor immune escape mechanisms do not impact pre-existing and consolidated immune responses, we tested the hypothesis of redirecting a pregenerated immunity to cancer: to recall a non-tumor antigen response against the tumor, silk fibroin nanoparticles (SFNs) have been selected as 'Trojan-horse' carriers, promoting the antigen uptake by the tumor cells. METHODS: SFNs have been loaded with either ovalbumin (OVA) or CpG oligonucleotide (CpG) as antigen or adjuvant, respectively. In vitro uptake of SFNs by tumor (B16/F10 melanoma and MB49 bladder cancer) or dendritic cells, as well as the presence of OVA-specific T cells in splenic and tumor-infiltrating lymphocytes, were assessed by cytometric analyses. Proof-of-concept of in vivo efficacy was achieved in an OVA-hyperimmune B16/F10 murine melanoma model: SFNs-OVA or SFNs-CpG were injected, separately or in association, into the subcutaneous peritumoral area. Cancer dimensions/survival time were monitored, while, at the molecular level, system biology approaches based on graph theory and experimental proteomic data were performed. RESULTS: SFNs were efficiently in vitro uptaken by cancer and dendritic cells. In vivo peritumor administration of SFNs-OVA redirected OVA-specific cytotoxic T cells intratumorally. Proteomics and systems biology showed that peritumoral treatment with either SFNs-OVA or SFNs-CpG dramatically modified tumor microenvironment with respect to the control (CTR), mainly involving functional modules and hubs related to angiogenesis, inflammatory mediators, immune function, T complex and serpins expression, redox homeostasis, and energetic metabolism. Both SFNs-OVA and SFNs-CpG significantly delayed melanoma growth/survival time, and their effect was additive. CONCLUSIONS: Both SFNs-OVA and SFNs-CpG induce effective anticancer response through complementary mechanisms and show the efficacy of an innovative active immunotherapy approach based on the redirection of pre-existing immunity against cancer cells. This approach could be universally applied for solid cancer treatments if translated into the clinic using re-call antigens of childhood vaccination.

Polyurethane and polyurethane/hydroxyapatite scaffold in a three-dimensional culture system.

Designing a new scaffold with an optimal ability of osteogenesis differentiation is a significant step bone tissue engineering along with the growing demands for bone craft in recent decades. Herein, we used Polyurethane (PU), a novel biocompatible and flexible polymer, and Hydroxyapatite (HA), the major component of human hard tissues matrix for developing new scaffolds and analyzing the in vitro osteogenic differentiation potential of human adipose-derived mesenchymal stem cells (Ad-MSCs) in basal and induction media. Gene expression analysis was performed to evaluate the expression level of four osteogenic differentiation genes. MTT assays were also done to assess the attachment and proliferation of the cells after 7 and 21 days of seeding to scaffolds. The expression level of RUNX2 was increased in seeded cells on PU/HA scaffolds compared with the PU. Cellular adhesion and proliferation of the Ad-MSCs were higher in PU/HA than PU scaffolds according to the histology analysis. The PU and PU/HA scaffolds supported the attachment, proliferation, and differentiation of Ad-MSCs, and they are suitable candidates for producing constructs in bone regeneration. However, further in-vitro and in-vivo studies on these scaffolds are needed to introduce an appropriate candidate for clinical bone regeneration.

Innovative Cell and Platelet Rich Plasma Therapies for Diabetic Foot Ulcer Treatment: The Allogeneic Approach.

Cutaneous chronic wounds are a major global health burden in continuous growth, because of population aging and the higher incidence of chronic diseases, such as diabetes. Different treatments have been proposed: biological, surgical, and physical. However, most of these treatments are palliative and none of them can be considered fully satisfactory. During a spontaneous wound healing, endogenous regeneration mechanisms and resident cell activity are triggered by the released platelet content. Activated stem and progenitor cells are key factors for ulcer healing, and they can be either recruited to the wound site from the tissue itself (resident cells) or from elsewhere. Transplant of skin substitutes, and of stem cells derived from tissues such as bone marrow or adipose tissue, together with platelet-rich plasma (PRP) treatments have been proposed as therapeutic options, and they represent the today most promising tools to promote ulcer healing in diabetes. Although stem cells can directly participate to skin repair, they primarily contribute to the tissue remodeling by releasing biomolecules and microvesicles able to stimulate the endogenous regeneration mechanisms. Stem cells and PRP can be obtained from patients as autologous preparations. However, in the diabetic condition, poor cell number, reduced cell activity or impaired PRP efficacy may limit their use. Administration of allogeneic preparations from healthy and/or younger donors is regarded with increasing interest to overcome such limitation. This review summarizes the results obtained when these innovative treatments were adopted in preclinical animal models of diabetes and in diabetic patients, with a focus on allogeneic preparations.

Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol.

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).

GMP-compliant sponge-like dressing containing MSC lyo-secretome: Proteomic network of healing in a murine wound model.

Chronic wounds account for 3% of total healthcare expenditure of developed countries; thus, innovative therapies, including Mesenchymal Stem Cells (MSCs) end their exosomes are increasingly considered, even if the activity depends on the whole secretome, made of both soluble proteins and extracellular vesicles. In this work, we prove for the first time the in vivo activity of the whole secretome formulated in a sponge-like alginate wound dressing to obtain the controlled release of bioactive substances. The product has been prepared in a public GMP-compliant facility by a scalable process; based on the murine model, treated wounds healed faster than controls without complications or infections. The treatment induced a higher acute inflammatory process in a short time and sustained the proliferative phase by accelerating fibroblast migration, granulation tissue formation, neovascularization and collagen deposition. The efficacy was substantially supported by the agreement between histological and proteomic findings. In addition to functional modules related to proteolysis, complement and coagulation cascades, protein folding and ECM remodeling, in treated skin, emerged the role of specific wound healing related proteins, including Tenascin (Tnc), Decorin (Dcn) and Epidermal growth factor receptor (EGFR). Of note, Decorin and Tenascin were also components of secretome, and network analysis suggests a potential role in regulating EGFR. Although further experiments will be necessary to characterize better the molecular keys induced by treatment, overall, our results confirm the whole secretome efficacy as novel "cell-free therapy". Also, sponge-like topical dressing containing the whole secretome, GMP- compliant and "ready-off-the-shelf", may represent a relevant point to facilitate its translation into the clinic.

Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.

Growth Factors Delivery System for Skin Regeneration: An Advanced Wound Dressing.

Standard treatments of chronic skin ulcers based on the direct application of dressings still present several limits with regard to a complete tissue regeneration. Innovative strategies in tissue engineering offer materials that can tune cell behavior and promote growth tissue favoring cell recruitment in the early stages of wound healing. A combination of Alginate (Alg), Sericin (SS) with Platelet Lysate (PL), as a freeze-dried sponge, is proposed to generate a bioactive wound dressing to care skin lesions. Biomembranes at different composition were tested for the release of platelet growth factors, cytotoxicity, protective effects against oxidative stress and cell proliferation induction. The highest level of the growth factors release occurred within 48 h, an optimized time to burst a healing process in vivo; the presence of SS differently modulated the release of the factors by interaction with the proteins composing the biomembranes. Any cytotoxicity was registered, whereas a capability to protect cells against oxidative stress and induce proliferation was observed when PL was included in the biomembrane. In a mouse skin lesion model, the biomembranes with PL promoted the healing process, inducing an accelerated and more pronounced burst of inflammation, formation of granulation tissue and new collagen deposition, leading to a more rapid skin regeneration.

Total disc replacement for lumbar degenerative disc disease: single centre 20 years experience.

PURPOSE: To report clinical and radiographic outcomes, rate of complications and influence on spinal alignment on long-term follow-up (FU) of patients who underwent lumbar total disc arthroplasty (TDR), bringing some evidence to determine the profile of the most well-suited patients for TDR. METHODS: A retrospective review of patients underwent TDR for low back pain from degenerative disc disease (DDD) resistant to conservative treatment was performed. Demographic features, surgical data, clinical and radiographic outcomes, complications and spinopelvic parameters were evaluated. RESULTS: Thirty patients (32 TDR) were included with a mean FU of 164 ± 36.5 months. The clinical outcomes measured by visual analogue scale and Oswestry Disability Index showed a significant improvement between preoperative and 1-year FU (p < 0.01). No significant temporal variance has been identified between 1-year and long-term follow-up (p > 0.05). The surgical revision rate was 10%. The overall rate of complications was 20%. At final follow-up, the mobility of the prosthesis was preserved in 68.75% of the cases, and 73.3% of the patients were globally well aligned. CONCLUSION: The optimal surgical indication is crucial to achieve excellent clinical and radiological outcomes. According to the literature and to our experience, we underline the importance of a coronal deformity < 15° Cobb angle and a Roussouly type 1 or 2 as the profile of the most well-suited patient for TDR. Our long-term results confirm the existing evidence about efficacy and safety of TDR as a reliable option, in optimal surgery indication, to treat DDD. These slides can be retrieved under Electronic Supplementary Material.

Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

: Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.

Synchrotron radiation techniques boost the research in bone tissue engineering.

X-ray Synchrotron radiation-based techniques, in particular Micro-tomography and Micro-diffraction, were exploited to investigate the structure of bone deposited in vivo within a porous ceramic scaffold. Bone formation was studied by implanting Mesenchymal Stem Cell (MSC) seeded ceramic scaffolds in a mouse model. Osteoblasts derived from the seeded MSC and from differentiation of cells migrated within the scaffold together with the blood vessels, deposited within the scaffold pores an organic collagenous matrix on which a precursor mineral amorphous liquid-phase, containing Ca++ and PO4-- crystallized filling the gaps between the collagen molecules. Histology offered a valid instrument to investigate the engineered tissue structure, but, unfortunately, limited itself to a macroscopic analysis. The evolution of the X-ray Synchrotron radiation-based techniques and the combination of micro X-ray diffraction with X-ray phase-contrast imaging enabled to study the dynamic of the structural and morphological changes occurring during the new bone deposition, biomineralization and vascularization. In fact, the unique features of Synchrotron radiation, is providing the high spatial resolution probe which is necessary for the study of complex materials presenting heterogeneity from micron-scale to meso- and nano-scale. Indeed, this is the occurrence in the heterogeneous and hierarchical bone tissue where an organic matter, such as the collagenous matrix, interacts with mineral nano-crystals to generate a hybrid multiscale biomaterial with unique physical properties. In this framework, the use of advanced synchrotron radiation techniques allowed to understand and to clarify fundamental aspects of the bone formation process within the bioceramic, i.e. biomineralization and vascularization, including to obtain deeper knowledge on bone deposition, mineralization and reabsorption in different health, aging and pathological conditions. In this review we present an overview of the X-ray Synchrotron radiation techniques and we provide a general outlook of their applications on bone Tissue Engineering, with a focus on our group work. STATEMENT OF SIGNIFICANCE: Synchrotron Radiation techniques for Tissue Engineering In this review we report recent applications of X-ray Synchrotron radiation-based techniques, in particular Microtomography and Microdiffraction, to investigations on the structure of ceramic scaffolds and bone tissue regeneration. Tissue engineering has made significant advances in bone regeneration by proposing the use of mesenchymal stem cells in combination with various types of scaffolds. The efficacy of the biomaterials used to date is not considered optimal in terms of resorbability and bone formation, resulting in a poor vascularization at the implant site. The review largely based on our publications in the last ten years could help the study of the regenerative model proposed. We also believe that the new imaging technologies we describe could be a starting point for the development of additional new techniques with the final aim of transferring them to the clinical practice.

In vitro efficacy of silk sericin microparticles and platelet lysate for intervertebral disk regeneration.

Intervertebral disk degeneration is an oxidative and inflammatory pathological condition that induces viability and functionality reduction of Nucleus Pulposus cells (NPs). Cellular therapies were previously proposed to repair and substitute the herniated disk but low proliferative index and pathological conditions of NPs dramatically reduced the efficacy of this approach. To overcome these problems we proposed, for the first time, a therapeutic system based on the association of silk sericin microparticles and platelet-derived products. Silk sericin (SS) is a bioactive protein with marked antioxidant properties, while platelet lysate (PL) and platelet poor plasma (PPP) represent a source of growth factors able to support cell viability and to promote tissue regeneration. We demonstrated that the mixture PL + PPP promoted NPs proliferation with a significant reduction of cellular doubling time. SS microparticles, alone or in combination with PPP, presented the higher ROS-scavenging activity while, SS microparticles and PL resulted as the best association able to protect NPs against oxidative stress induce by hydroxide peroxide. Based on these results, the authors are confident that, with the ever increasing need of efficacious tools for regenerative medicine purposes, SS microparticles and PL + PPP association could represent an effective approach for the development of low impact and non-invasive therapies.

Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. STATEMENT OF SIGNIFICANCE: Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and store them, resulting in an easy and fast accessibility and an expanded use of hPL for wound healing.

Platelet-rich plasma-based bioactive membrane as a new advanced wound care tool.

Chronic skin ulcers, consequence of diabetes and other pathological conditions, heavily compromise the patient life quality and represent a high and constantly growing cost for National Health Services. Autologous platelet-rich plasma (PRP), has been proposed to treat these lesions. The absence of guidelines for the PRP production and the need of a fresh preparation for each treatment lead us to develop a protocol for the production of an allogenic PRP-based bioactive membrane (BAM), standardized for platelet concentration and growth factor release. This work compares BAMs obtained starting from two different platelet concentrations. There was no direct correlation between the amount of growth factors released by BAM in vitro and the initial platelet count. However, different release kinetics were noticed for different growth factors, suggesting that they were differently retained by the two BAMs. The angiogenic potential of both BAMs was determined by Luminex Angiogenesis Assay. The biological activity of the factors released by the two BAMs was confirmed by cell proliferation and migration. A diabetic mouse chronic ulcer model was used to define the best PRP therapeutic dose in vivo. Both BAMs induced wound healing by increasing the thickness of the regenerated epidermis and the vessel number. However, a too high platelet concentration resulted in a slowdown of the membrane resorption that interfered with the skin healing. Overall, the results indicate that the BAMs could represent a natural and effective wound healing tool for the treatment of skin ulcers. Copyright © 2016 John Wiley & Sons, Ltd.

Allogeneic platelet-rich plasma affects monocyte differentiation to dendritic cells causing an anti-inflammatory microenvironment, putatively fostering wound healing.

Autologous platelet-rich plasma (PRP) is used clinically to induce repair of different tissues through the release of bioactive molecules. In some patients, the production of efficient autologous PRP is unfeasible due to their compromised health. Allogeneic PRP mismatched for AB0 and Rh antigens was developed. The effect of allogeneic PRP on immune response should be defined to use it in clinical practice avoiding side effects. Thus, whether PRP affects the differentiation of peripheral blood monocytes to dendritic cells upon stimulation with granulocyte monocyte colony stimulating factor and interleukin-4 was investigated. Indeed, these cells are the main players of immune response and tissue repair. PRP inhibited the differentiation of monocytes to CD1a+ dendritic cells and favoured the expansion of phagocytic CD163+ CD206+ fibrocyte-like cells. These cells produced interleukin-10 and prostaglandin-E2 , but not interferon-γ, upon stimulation with lipopolysaccharides. Moreover, they promoted the expansion of regulatory CD4+ CD25+ FoxP3+ T cells upon allostimulation or antigen specific priming. Finally, the conditioned medium harvested from monocytes differentiated with PRP triggered a strong chemotactic effect on mesenchymal cells in both scratch and transwell migration assays. These results strongly suggest that allogeneic PRP can foster the differentiation of monocytes to a regulatory anti-inflammatory population, possibly favouring wound healing. Copyright © 2016 John Wiley & Sons, Ltd.

Platelet Lysate Activates Human Subcutaneous Adipose Tissue Cells by Promoting Cell Proliferation and Their Paracrine Activity Toward Epidermal Keratinocytes.

Skin chronic wounds are non-healing ulcerative defects, which arise in association with a morbidity state, such as diabetes and vascular insufficiency or as the consequence of systemic factors including advanced age. Platelet Rich Plasma, a platelet-rich blood fraction, can significantly improve the healing of human skin chronic ulcers. Given that the subcutaneous adipose tissue is located beneath the skin and plays a role in the skin homeostasis, in this study, we investigated the in vitro response of human subcutaneous adipose tissue cells to platelet content in a model mimicking in vitro the in situ milieu of a deep skin injury. Considering that, at the wound site, plasma turn to serum, platelets are activated and inflammation occurs, human adipose-derived stromal cells (hASC) were cultured with Human Serum (HS) supplemented or not with Platelet Lysate (PL) and/or IL-1α. We observed that HS sustained hASC proliferation more efficiently than FBS and induced a spontaneous adipogenic differentiation in the cells. PL added to HS enhanced hASC proliferation, regardless the presence of IL-1α. In the presence of PL, hASC progressively lessened the adipogenic phenotype, possibly because the proliferation of less committed cells was induced. However, these cells resumed adipogenesis in permissive conditions. Accordingly, PL induced in quiescent cells activation of the proliferation-related pathways ERK, Akt, and STAT-3 and expression of Cyclin D1. Moreover, PL induced an early and transient increase of the pro-inflammatory response triggered by IL-1α, by inducing COX-2 expression and secretion of a large amount of PGE2, IL-6, and IL-8. Media conditioned by PL-stimulated hASC exerted a chemotactic activity on human keratinocytes and favored the healing of an in vitro scratch wound. In order to bridge the gap between in vitro results and possible in vivo events, the stimuli were also tested in ex vivo cultures of in toto human adipose tissue biopsies (hAT). PL induced cell proliferation in hAT and outgrowth of committed progenitor cells able to differentiate in permissive conditions. In conclusion, we report that the adipose tissue responds to the wound microenvironment by activating the proliferation of adipose tissue progenitor cells and promoting the release of factors favoring wound healing.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

AIM: This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. MATERIALS & METHODS: Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. RESULTS & CONCLUSION: Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

Learning from Mother Nature: Innovative Tools to Boost Endogenous Repair of Critical or Difficult-to-Heal Large Tissue Defects.

For repair of chronic or difficult-to-heal tissue lesions and defects, major constraints exist to a broad application of cell therapy and tissue engineering approaches, i.e., transplantation of "ex vivo" expanded autologous stem/progenitor cells, alone or associated with carrier biomaterials. To enable a large number of patients to benefit, new strategies should be considered. One of the main goals of contemporary regenerative medicine is to develop new regenerative therapies, inspired from Mother Nature. In all injured tissues, when platelets are activated by tissue contact, their released factors promote innate immune cell migration to the wound site. Platelet-derived factors and factors secreted by migrating immune cells create an inflammatory microenvironment, in turn, causing the activation of angiogenesis and vasculogenesis processes. Eventually, repair or regeneration of the injured tissue occurs via paracrine signals activating, mobilizing or recruiting to the wound site cells with healing potential, such as stem cells, progenitors, or undifferentiated cells derived from the reprogramming of tissue differentiated cells. This review, largely based on our studies, discusses the identification of new tools, inspired by cellular and molecular mechanisms overseeing physiological tissue healing, that could reactivate dormant endogenous regeneration mechanisms lost during evolution and ontogenesis.

A humanized system to expand in vitro amniotic fluid-derived stem cells intended for clinical application.

BACKGROUND AIMS: The amniotic fluid is a new source of multipotent stem cells with therapeutic potential for human diseases. In agreement with the regulatory requirement to reduce and possibly to avoid animal-derived reagents in the culture of cells intended for cell therapy, bovine serum, the most common supplement in the culture medium, was replaced by human platelet-derived growth factors. METHODS: We tested a new culture medium to expand monolayers of human amniotic fluid stem cells (hAFSC) for clinical use. The AFSC were isolated by c-Kit selection and expanded in media supplemented with either bovine serum or a human platelet lysate (Lyset). RESULTS: We compared proliferation kinetics, colony-forming unit percentage, multilineage differentiation, immunophenotypic characterization and inhibition of peripheral blood mononuclear cell proliferation of the two AFSC cell cultures and we found no significant differences. Moreover, the karyotype analysis of the cells expanded in the presence of the platelet lysate did not present cytogenetic abnormalities and in vitro and in vivo studies revealed no cell tumorigenicity. CONCLUSIONS: Platelet derivatives represent a rich source of growth factors that can play a safety role in the homeostasis, proliferation and remodeling of tissue healing. We propose human platelet extracts as a preferential alternative to animal serum for the expansion of stem cells for clinical applications.