Enrico Lucarelli

Platelet-derived growth factors enhance proliferation of human stromal stem cells.

Studies on new procedures for bone reconstruction suggest that autologous cells seeded on a resorbable scaffold can improve the treatment of bone defects. It is important to develop culture conditions for ex vivo expansion of stromal stem cells (SSC) that do not compromise their self-renewing and differentiation capability. Bone marrow SSC and platelet gel (PG) obtained by platelet-rich plasma provide an invaluable source for autologous progenitor cells and growth factors for bone reconstruction. In this study the effect of platelet-rich plasma (PRP) released by PG on SSC proliferation and differentiation was investigated. MTT assay was used to investigate the effect of PRP on proliferation: results showed that PRP induced SSC proliferation. The effect was dose dependent and 10% PRP is sufficient to induce a marked cell proliferation. Untreated cells served as controls. Upon treatment with 10% PRP, cells entered logarithmic growth. Removal of PRP restored the characteristic proliferation rate. Because SSC can gradually lose their capability to differentiate along the chondrogenic and osteogenic lineage during subculture in vitro, we tested whether 10% PRP treatment affected SSC ability to mineralize. SSC were first exposed to 10% PRP for five passages, at passage 6 PRP was washed away and plated cells were treated with dexamethasone (DEX). DEX induced a three-fold increase in the number of alkaline phosphatase positive cells and induced mineralization that is consistent with the differentiation of osteochondroprogenitor cells. In conclusion, 10% PRP promotes SSC proliferation; cells expanded with 10% PRP can mineralize the extracellular matrix once PRP is withdrawn.

CTLA‐4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction

CTLA‐4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA‐4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA‐4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast‐like cultures). However, by reverse transcriptase‐PCR, CTLA‐4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA‐4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA‐4‐expressing tumor lines with recombinant forms of the CTLA‐4‐ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase‐8 and caspase‐3. The level of apoptosis was reduced by soluble CTLA‐4 and by anti‐CTLA‐4 scFvs antibodies. The novel finding that CTLA‐4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.

Stromal stem cells and platelet-rich plasma improve bone allograft integration.

Early vascular invasion is a key factor in bone allograft incorporation. It may reduce the complications related to slow and incomplete bone integration. Bone-marrow-derived stromal stem cells associated with platelet-rich plasma are potent angiogenic inducers proven to release vascular endothelial growth factor. Our goal was to test whether the combination of stromal stem cells and platelet-rich plasma is able to increase massive allograft integration in a large animal model with sacrifice at 4 months. A critical defect was made in the mid-diaphysis of the metatarsal bone of 10 sheep; the study group received an allograft plus stromal stem cells, platelet-rich plasma, and collagen (six animals) and the control group received only the allograft (four animals). Investigation was done with radiographs, mechanical tests and histomorphometric analysis, including new vascularization. Results showed substantial new bone formation in the allograft of the study group. Bone formation is correlated with better vascular invasion and remodeling of the graft in the study group. These results confirm the key role played by stromal stem cells and platelet-rich plasma in bone repair. Further studies are needed to better define the role stromal stem cells play when implanted alone.

Ultrastructural Characteristics of Human Mesenchymal Stromal (Stem) Cells Derived from Bone Marrow and Term Placenta

Human mesenchymal stromal (stem) cells (hMSCs) isolated from adult bone marrow (BM-hMSCs) as well as amnion (AM-hMSCs) and chorion (CM-hMSCs) term placenta leaves were studied by transmission electron microscopy (TEM) to investigate their ultrastructural basic phenotype. At flow cytometry, the isolated cells showed a homogeneous expression of markers commonly used to identify hMSCs, i.e., CD105, CD44, CD90, CD166, HLA-ABC positivities, and CD45, AC133, and HLA-DR negativities. However, TEM revealed subtle yet significant differences. BM-hMSCs had mesenchymal features with dilated cisternae of rough endoplasmic reticulum (rER) and peripheral collections of multiloculated clear blisters; this latter finding mostly representing complex foldings of the plasma membrane could be revelatory of the in situ cell arrangement in the niche microenvironment. Unlike BM-hMSCs, CM-hMSCs were more primitive and metabolically quiescent, their major features being the presence of rER stacks and large peripheral collections of unbound glycogen. AM-hMSCs showed a hybrid epithelial–mesenchymal ultrastructural phenotype; epithelial characters included non-intestinal-type surface microvilli, intracytoplasmic lumina lined with microvilli, and intercellular junctions; mesenchymal features included rER profiles, lipid droplets, and well-developed foci of contractile filaments with dense bodies. These features are consistent with the view that AM-hMSCs have a pluripotent potential. In conclusion, this study documents that ultrastructural differences exist among phenotypically similar hMSCs derived from human bone marrow and term placenta leaves; such differences could be revelatory of the hMSCs in vitro differentiation potential and may provide useful clues to attempt their in situ identification.

Effect of Mesenchymal Stem Cells and Platelet-Rich Plasma on the Healing of Standardized Bone Defects in the Alveolar Ridge: A Comparative Histomorphometric Study in Minipigs

PURPOSE The purpose of this study was to test the effect of the combination of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) incorporated into a fluorohydroxyapatite (FHA) scaffold on bone regeneration in cylindrical defects in the edentulous mandibular ridge of minipigs. MATERIALS AND METHODS Two mandibular premolar teeth were extracted bilaterally in 8 adult minipigs. After 2 months, 4 standardized defects of 3.5 mm diameter and 8 mm depth were created in each root site. The defects were randomly grafted with autogenous mandibular bone, FHA alone, PRP-FHA, or MSCs-PRP-FHA. A resorbable collagen membrane was placed over the defect area and the flaps were sutured. The animals were sacrificed 3 months later and biopsy samples were taken from the defect sites for histologic and histomorphometric assessment. RESULTS There was no evidence of inflammation or adverse tissue reaction with either treatment. MSCs-PRP-FHA-treated sites showed new vital bone between residual grafting particles. PRP-FHA- and FHA-treated sites showed residual particles in a background of marrow soft tissue with a moderate quantity of newly formed bone. Autogenous bone (46.97%) and MSCs-PRP-FHA (45.28%) produced a significantly higher amount of vital bone than PRP-FHA (37.95%), or FHA alone (36.03%). Further, the MSCs-PRP-FHA-treated defects showed a significantly higher percentage of contact between graft particles and newly formed bone compared with PRP-FHA and FHA group (59.23% vs 48.37% and 46.43%, respectively). CONCLUSIONS Our results suggest that, in this animal model, the addition of MSCs to PRP-FHA enhances bone formation after 3 months.

Dose-dependent effect of adipose-derived adult stem cells on vertical bone regeneration in rabbit calvarium.

Previous in vivo studies have shown a limited potential for vertical bone regeneration using osteoconductive scaffolds alone. In the present study, we investigated whether the association of adipose-derived adult stem cells (ASCs) with anorganic bovine bone (ABB) scaffold improved bone formation and implant osseointegration in a vertical guided bone regeneration model. Two pre-formed titanium domes were placed on the calvaria of 12 rabbits. Four treatment modalities were evenly distributed among the 24 domes: ABB alone, and ABB containing 3 x 10(5), 3 x 10(6), or 3 x 10(7) cells/graft. After 1 month, the domes were removed and one titanium implant was placed into each augmented site. One month after the second operation, the animals were killed and biopsy specimens were examined by histomorphometric and micro-CT analyses. Results indicated that at all concentrations, the ASC-loaded groups showed significantly more new bone formation and higher mean values of bone-implant contact and bone density inside threads than the ABB group. Furthermore, ASCs demonstrated a dose-response relationship, with the highest dose chosen inducing more robust bone regeneration. This study suggests that the delivery of ASCs on ABB might effectively increase vertical bone regeneration and implant osseointegration, versus ABB alone.

Prognostic Value of CCN3 in Osteosarcoma

Purpose: Osteosarcoma, the most common bone tumor, lacks prognostic markers that could distinguish patients before therapy and drive treatment choices. We assessed the prognostic value of CCN1, CCN2, and CCN3 genes, involved in fundamental biological processes. Experimental Design: Expression of CCN1, CCN2, and CCN3 was measured by quantitative PCR in 45 newly diagnosed osteosarcomas. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations with osteoblastic differentiation and/or drug response genes were assessed in tumor cells using Spearman correlation and Fishers exact tests. Results:CCN1 and CCN2 expression was associated with genes involved in commitment of mesenchymal stem cells toward osteoblasts and in early phases of osteoblastic differentiation (RUNX family genes; cadherin 4, 11, and 13; jun and fos; collagen I and SPARC). Although CCN3 is barely expressed in normal proliferating osteoblasts and mesenchymal stem cells, its expression was generally high in osteosarcoma and its level of expression did not correlate with any specific osteoblastic differentiation genes. High expression of CCN3 significantly correlated with worse prognosis in osteosarcoma. This may be only partly explained by the association with the expression of multidrug resistance–related protein 1 and 4, two ATP-binding cassette transporters that also acted as predictors of worse outcome in our study. Conclusions: Our study showed temporal and coordinated expression of CCN1, CCN2, and CCN3 genes during osteoblastic differentiation and highlighted significant differences between human normal and osteosarcoma cell differentiation in vitro. CCN1 and CCN2 expression shows no prognostic relevance in osteosarcoma. In contrast, assessment for CCN3 expression levels at diagnosis may represent a useful molecular tool to early identification of patients with different prognosis.

Presence of telomerase activity in different musculoskeletal tumor histotypes and correlation with aggressiveness.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. Telomerase activity was observed and correlated with aggressiveness in different neoplasms such as breast, prostate, blood and brain cancers, among others. To investigate whether telomerase activity is an index of aggressiveness in bone and soft tissue lesions of the extremities, 66 biopsy samples from our tissue bank were studied. These samples included 43 high‐grade sarcomas, 9 aggressive benign tumors and 14 totally benign lesions. The samples were collected from patients homogeneously treated at the Rizzoli Orthopaedic Institute with a follow‐up ranging from 4 to 11 years (median, 7 years). A non‐radioactive polymerase chain reaction‐based enzyme‐linked immunosorbent assay was used for the study. All tumors investigated were positive for telomerase activity. Among benign lesions, only 2 aneurysmal bone cysts showed higher telomerase activity than the cut‐off point, whereas all the other benign lesions had lower activity. Our results indicate that high levels of telomerase activity in bone and soft tissue lesions correlate with more aggressive clinical behavior in patients treated with surgery alone. An interesting inverse correlation between telomerase activity and occurrence of pulmonary metastasis was detected in osteosarcoma patients treated with chemotherapy. A parallel increase of telomerase activity and malignancy was observed in the adipose and cartilagineous tissue lesions. Our data suggest that telomerase activity could be considered a marker of tumor aggressiveness for bone and soft tissue lesions. The results obtained in osteosarcoma samples suggest that low levels of telomerase activity may be predictive of the prognosis and should influence the therapeutic protocol.

Comparison of alternative mesenchymal stem cell sources for cell banking and musculoskeletal advanced therapies

With the continuous discovery of new alternative sources containing mesenchymal stem cells (MSCs), regenerative medicine therapies may find tailored applications in the clinics. Although these cells have been demonstrated to express specific mesenchymal markers and are able to differentiate into mesenchymal lineages in ad hoc culture conditions, it is still critical to determine the yield and differentiation potential of these cells in comparative studies under the same standardized culture environment. Moreover, the opportunity to use MSCs from bone marrow (BM) of multiorgan donors for cell banking is of relevant importance. In the attempt to establish the relative potential of alternative MSCs sources, we analyzed and compared the yield and differentiation potential of human MSCs from adipose and BM tissues of cadaveric origins, and from fetal annexes (placenta and umbilical cord) after delivery using standardized isolation and culture protocols. BM contained a significantly higher amount of mononuclear cells (MNCs) compared to the other tissue sources. Nonetheless, a higher cell seeding density was needed for these cells to successfully isolate MSCs. The MNCs populations were highly heterogeneous and expressed variable MSCs markers with a large variation from donor to donor. After MSCs selection through tissue culture plastic adhesion, cells displayed a comparable proliferation capacity with distinct colony morphologies and were positive for a pool of typical MSCs markers. In vitro differentiation assays showed a higher osteogenic differentiation capacity of adipose tissue and BM MSCs, and a higher chondrogenic differentiation capacity of BM MSCs. J. Cell. Biochem. 112: 1418–1430, 2011.

Mesenchymal stem cells and platelet-rich plasma enhance bone formation in sinus grafting: a histomorphometric study in minipigs

OBJECTIVES Autologous, allogenic, and alloplastic materials for sinus augmentation have specific drawbacks, which has stimulated an ongoing search for new materials and tissue-engineering constructs. We investigated whether mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) seeded on a fluorohydroxyapatite (FH) scaffold can improve bone formation and bone-to-implant contact (BIC) in maxillary sinus grafting. MATERIAL AND METHODS Bilateral sinus augmentation procedures were performed in eight minipigs. MSCs, PRP, and FH scaffold (test site) or FH alone (control site) were grafted in each maxillary sinus. Distal to the osteotomy, one dental implant per sinus was placed in the grafting material through the facial sinus wall. The animals were killed 3 months after grafting, and block sections of the implant sites were harvested and prepared for histomorphometric analysis. RESULTS After 12 weeks, a significant increase in bone formation occurred in the test sites compared with the control sites (42.51%versus 18.98%; p=0.001). In addition, BIC was significantly greater in the test sites compared with the control sites in the regenerated area (23.71%versus 6.63%; p=0.028). CONCLUSIONS These findings show that sinus augmentation with MSCs-PRP, combined with FH may enhance bone formation and osseointegration of dental implants compared with FH alone in minipigs.