Paolo Strada

Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

The heterogeneous population of non‐haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet‐rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet‐derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro‐expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue‐engineering applications. Copyright

Biological activity of a standardized freeze-dried platelet derivative to be used as cell culture medium supplement.

Abstract Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in “traditional” parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an “off-the-shelf” product to be used for the selection and expansion of several cell types also in critical cell culture conditions.

Combined platelet and plasma derivatives enhance proliferation of stem/progenitor cells maintaining their differentiation potential

BACKGROUND AIMS Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. METHODS A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. RESULTS The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). CONCLUSIONS The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest.

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.

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

Regenerative surgery for the definitive surgical repair of enterocutaneous fistula.

1. Green MF, Gibson JR, Bryson JR, Thomson E. A one-stage correction of mandibular defects using a split sternum pectoralis major osteo-musculocutaneous transfer. Br J Plast Surg. 1981;34:11–16. 2. Robertson GA. A comparison between sternum and rib in osteomyocutaneous reconstruction of major mandibular defects. Ann Plast Surg. 1986;17:421–433. 3. Robertson GA. The role of sternum in osteomyocutaneous reconstruction of major mandibular defects. Am J Surg. 1986; 152:367–370. 4. Ariyan S. The pectoralis major myocutaneous flap: A versatile flap for reconstruction in the head and neck. Plast Reconstr Surg. 1979;63:73–81. Regenerative Surgery for the Definitive Surgical Repair of Enterocutaneous Fistula Sir: R surgery is a relatively new branch of surgery, born in 1997, when Whitman et al.1 proposed to integrate platelet-enriched plasma into fibrin glue. It is based on the use of stem cells and/or biological products (platelet-rich plasma or its gel formulation, platelet gel) that induce stem cell migration to the damaged tissues, stimulating their proliferation and eventually accomplishing tissue repair. Tissue repair is a complex biological process facilitated by growth factors. In addition to their functions in hemostasis, platelet -granules release several growth factors that promote tissue regeneration.2 Platelet gel is easy and safe to use. Addition of cryoprecipitate, thrombin, and Ca to platelet-rich plasma triggers the coagulation cascade, eventually resulting in the formation of a thrombus-like gelatinous substance. Growth factors are released following the platelet aggregation process.3 We describe the case of a 43-year-old woman who underwent several surgical interventions for endometriosis and presented a nonhealing ileocutaneous fistula. The first surgical intervention (June of 2004) consisted of endometriotic ovarian cyst and rectum-vaginal septum nodule removal and 24-cm rectum-sigmoid resection, complicated by a rectum-vaginal fistula, treated with left lateral colostomy. In January of 2008, a urologic computed tomographic scan documented bilateral hydronephrosis and a 4-cm endometriotic ovarian nodule: bilateral ureteral reimplantation with left vesical suspension and right tubo-ovarian resection were performed. Postoperative stay was complicated by a left sigmoidovesical fistula, treated with lateral colostomy, and an enterocutaneous fistula, treated conservatively with total parenteral nutrition. In April, endoscopic positioning of absorbable clips was performed to close the colovesical fistula edges. The patient came to our attention in June of 2010 still presenting the enterocutaneous fistula (Fig. 1). Each attempt to treat it had failed; thus, a regenerative surgical intervention using autologous platelet gel was scheduled. Autologous platelet concentrate, cryoprecipitate, and thrombin were obtained from 450 ml of whole blood. Platelet gel was prepared by adding 1 cc of autologous thrombin and then 1 cc of calcium gluconate to every 10 ml of platelet concentrate/cryoprecipitate solution. En bloc excision of the fistula tracks and surrounding fat tissue surrounding the fistulous tracks was performed: a first layer of platelet gel was positioned above the muscular band (Fig. 2), and a second layer was positioned to fill the superficial cavity between the fibroadipose flap and subcutaneous tissue. A single passive drain was positioned. Postoperative stay Fig. 3. The native mandible was burred down to create a step-

Regenerative Surgery for the Definitive Repair of a Vasculitic Nonhealing Ulcer Using Platelet-derived Growth Factors and Noncultured Autologous Cell Suspension.

Summary: Vasculitic ulcers are caused by numerous disorders and may be chronic if not well treated. Various modalities of treatment, both medical and surgical, are available. We describe the case of a 63-year-old patient with a vasculitic ulcer treated with platelet-derived growth factors and noncultured autologous cell suspension collected by an innovative single-use device (ReCell).