Anita Muraglia

Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

Regeneration of large bone defects in sheep using bone marrow stromal cells

Bone repair was addressed in a critical‐sized defect model in sheep, combining a ceramic biomaterial and mesenchymal progenitor cells. The defects in the tibial mid‐diaphysis were treated with autologous bone or with a silicon‐stabilized tricalcium phosphate biomaterial, implemented or not by the addition of expanded bone marrow stromal cells. An internal locking compression plate and an external fixator were applied for stabilization. Radiographies were taken during the 8 months follow‐up: the pixel grey levels of the lesion areas were determined to evaluate the repair process radiologically. Microradiography, histology and vascular density tests were performed. The autologous bone‐treated group performed best, as assessed radiologically, within 20–24 weeks after surgery. Very limited healing was detected in the other experimental group: a partial bone deposition occurred at the periphery of the bony stumps only in the cell‐seeded scaffolds. Interestingly, this effect ended within 20–24 weeks, as for the autologous bone, suggesting similar kinetics of the repair processes involved. Moreover, bone deposition was located where a significant reduction of the ceramic scaffold was detected. Faxitron microradiography and histology data confirmed these results. Vascular density analysis evidenced that cell‐seeded scaffolds supported an increased vascular ingrowth. Thus, the interactions with the proper microenvironment and the oxygen and nutrient supply in the inner part of the constructs seem fundamental to initiate scaffold substitution and to improve cell performance in tissue‐engineered approaches to bone repair. Copyright

Prefabricated engineered bone flaps: An experimental model of tissue reconstruction in plastic surgery

&NA; In light of the recently described experimental technique of in vivo bone reconstitution with biotechnologic methods (from bone marrow stromal cells) and the prefabrication flap procedures, the possibility to obtain autologous bone growth in a myocutaneous flap, thus creating a composite osteomyocutaneous preformed flap, is postulated. Human bone marrow stromal cells were delivered into the latissimus dorsi of athymic mice by a porous hydroxyapatite ceramic model. Eight weeks after the implantation, histologic examination revealed the presence of spongious bone tissue. A simple myocutaneous flap was thus transformed into a composite osteomyocutaneous flap. This flap is called the biotechnologic prefabricated flap, because it was the result of ex vivo expanded osteogenic precursor cells and in vivo bone tissue neoformation. The shape of the bone flap was exactly the same as the shape of the ceramic model used. A possible clinical application may be the correction of skeletal defects. The advantages of this procedure are simple surgical execution, the possibility of preshaping the graft to the exact characteristics of the defect, and the availability of autogenous donor tissue without donor site morbidity.

Microenvironment and stem properties of bone marrow- derived mesenchymal cells

Adult stem cells are self‐renewing, pluripotent, and able to repopulate the tissue in which they reside. Cells endowed with these properties have been isolated from several tissues and an increasing number of reports provide evidence of their ability, following transplantation, to engraft host tissues other than those of their origin. In this setting, interest in the well‐documented capacity of bone marrow stromal cells to undergo multilineage differentiation is growing. Neural and cardiomyogenic lineages have recently been proposed as additional differentiative pathways of these cells. However, culture conditions and inductive molecules can alter the behavior of bone marrow stromal cells and the microenvironment is critical for proper in vivo delivery. The maintenance of their stem properties and the possibility of reprogramming their commitment is a field of primary interest given the potential use of these cells in regenerative medicine. We discuss here how the microenvironmental cues, and the growth factors that physiologically govern commitment and subsequent differentiation, influence the properties of bone marrow stromal cells and modulate their engraftment into host tissues.

Fluorescence microscopy imaging of bone for automated histomorphometry.

We have developed a computer-based method for the automated quantification of bone tissue in histological sections of decalcified specimens. Bone tissue was generated by ectopic implantation of ceramic-based carriers loaded with human bone marrow stromal cells (BMSCs). The method is based on the acquisition of multimodal images, in order to identify and measure the area covered by bone tissue (using fluorescent light) and the total area of tissue (using transmitted light), thereby excluding the regions corresponding to nonresorbed scaffold. The amount of bone as a percentage of the total area of interest (bone/area) and of the newly formed tissue (bone/tissue) is automatically derived. The computer-based results correlated closely with those obtained by manual identification of bone and tissue areas in the same histological fields (R(2) = 0.997; p < 0.0005), with errors dependent on the magnification used but always lower than 9.4%. The method was used to compare the bone/tissue and bone/area percentages in samples of engineered bone based on human BMSCs expanded in the presence of different biochemical factors and loaded onto different scaffolds. The technique thus represents a valuable tool to quantify reproducibly, accurately, and easily bone formation in a variety of tissue-engineering studies.

Osteogenic differentiation of human mesenchymal stromal cells on surface-modified titanium alloys for orthopedic and dental implants.

Purpose Surface properties of titanium alloys, used for orthopedic and dental applications, are known to affect implant interactions with host tissues. Osteointegration, bone growth and remodeling in the area surrounding the implants can be implemented by specific biomimetic treatments; these allow the preparation of micro/nanostructured titanium surfaces with a thickened oxide layer, doped with calcium and phosphorus ions. We have challenged these experimental titanium alloys with primary human bone marrow stromal cells to compare the osteogenic differentiation outcomes of the cells once they are seeded onto the modified surfaces, thus simulating a prosthetic device-biological interface of clinical relevance. Methods A specific anodic spark discharge was the biomimetic treatment of choice, providing experimental titanium disks treated with different alkali etching approaches. The disks, checked by electron microscopy and spectroscopy, were subsequently used as substrates for the proliferation and osteogenic differentiation of human cells. Expression of markers of the osteogenic lineage was assessed by means of qualitative and quantitative PCR, by cytochemistry, immunohistochemistry Western blot and matrix metalloprotease activity analyses. Results Metal surfaces were initially less permissive for cell growth. Untreated control substrates were less efficient in sustaining mineralized matrix deposition upon osteogenic induction of the cells. Interestingly, bone sialo protein and matrix metalloprotease 2 levels were enhanced on experimental metals compared to control surfaces, particularly for titanium oxide coatings etched with KOH. Discussion As a whole, the KOH-modification of titanium surfaces seems to allow the best osteogenic differentiation of human mesenchymal stromal cells, representing a possible plus for future clinical prosthetic applications.

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.

DLX5 overexpression impairs osteogenic differentiation of human bone marrow stromal cells

The transcription factor DLX5 belongs to a family of homeoproteins required for craniofacial morphogenesis and forebrain development. DLX5 is expressed during formation of several skeletal elements such as cartilage, teeth and bone, and its knockout causes severe craniofacial malformations with a delay in the ossification process. Bone marrow contains mesenchymal progenitor cells which may differentiate along multiple pathways, therefore representing an interesting in vitro and in vivo model to study the mesodermal lineage differentiation. Here we report the effect of DLX5 overexpression in ex vivo expanded human bone marrow stromal cells by retroviral infection on the osteogenic lineage differentiation. A reduced mineral deposition was observed in DLX5-transduced cells upon osteogenic induction in culture. When DLX5-transduced cells were implanted in immunodeficient mice, a 60% reduction in bone matrix deposition was observed, whereas the in vitro chondrogenic potential was unaffected. A quantitative gene expression study indicated that DLX5 overexpression does not affect the early osteogenic commitment of bone marrow stromal cells but prevents their terminal differentiation. This block may be mediated by the observed persistent expression of SOX2, a transcription factor known to inhibit osteogenic differentiation.

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.