**3. Purification and culture**

The current methods and techniques employed for the isolation and characterization of peripheral blood fibrocytes are based mainly in the derivation of these cells from the buffy coat of peripheral blood obtained from human or animal sources. Circulating fibrocytes comprise the ~0.1-0.5% of the non-erythrocytic cells in the peripheral blood and they can be quantified and analyzed by flow cytometry (Bucala et al., 1994, Moeller et al., 2009).

monocytic fibroblast precursor was first proposed more than a hundred years ago by James Paget, and probably represents the first observations of cells with the molecular features of circulating fibrocytes (Herzog & Bucala 2010). Afterward in the early 1960´s the hypothesis of the blood borne origin of fibroblast appeared again in the literature; of particular significance are the observations of Petrakis and co-workers who reported the in vivo differentiation of human leukocytes into fibroblasts, histiocytes and adipocytes in subcutaneous diffusion chambers (Petrakis et al., 1961). More recently, it was demonstrated that bone-marrow (BM) contributes to the expansion of the broblast population in multiple organs and tissues, including skin, stomach and esophagus using mouse transplantation models, and in human liver fibrosis (Direkze et al 2003, 2004 and Forbes et al 2004). Regarding the lung, a pioneer work published in 2004 described that the collagen-producing broblasts in experimental pulmonary brosis are derived from BM progenitor cells (Hashimoto et al., 2004). While these studies documented the BM origin of at least part of the tissue broblasts during injury, they did not resolve whether these BM derived broblasts were from hematopoietic stem cells (HSCs) or mesenchymal stem cells. Later, through a model of transplantation of clones of cells derived from a single HSC from transgenic enhanced green uorescent protein (EGFP) mice, it

was clearly demonstrated that brocytes are derived from HSCs (Ebihara et al., 2006).

and to name a cell constituent of the inner ear spiral ligament, (Quan et al., 2004).

Sjöland et al., 2008; El-Asrar et al., 2008; Strieter et al., 2009).

**3. Purification and culture** 

Now it is known that fibrocytes are a hematopoietic stem cell source of fibroblasts/myofibroblasts that participate in the mechanisms of wound healing and fibrosis in many organs (Schmidt et al., 2003; Mori et al., 2005; Ebihara et al., 2006; Andersson-

The current methods and techniques employed for the isolation and characterization of peripheral blood fibrocytes are based mainly in the derivation of these cells from the buffy coat of peripheral blood obtained from human or animal sources. Circulating fibrocytes comprise the ~0.1-0.5% of the non-erythrocytic cells in the peripheral blood and they can be

quantified and analyzed by flow cytometry (Bucala et al., 1994, Moeller et al., 2009).

The circulating fibrocyte was first described in 1994 by Bucala, in a model of wound healing response, with the surgical implantation of wound chambers into the subcutaneous tissues of mice. The implantation resulted in a rapid influx of peripheral blood cells such as neutrophils, monocytes, and lymphocyte subpopulations within 24 hr. They noticed that 10% of the cells present in the wound chamber, were spindle shaped cells and expressed collagen I, and CD34, (Bucala et al., 1994). The idea that these cells were of circulating origin arose from the observation that their arrival in to the wound chamber was much faster than would be expected by entry of fibroblasts from the surrounding tissue, since the fibroblasts would have to migrate across the permeable plastic layer, enter the wound chamber, and begin matrix deposition, (Bucala, 2008). Hence, the entrance of large numbers of fibroblastlike cells simultaneously with circulating inflammatory cells suggested that this cell population was from peripheral blood origin and not exclusively by slow migration from adjacent connective tissue (Bucala et al., 1994). This new leukocyte sub-population was termed "brocytes", which combines the greek "kytos" referring to cell, and "bro", which is from the latin denoting ber. This nomenclature may lead to some overlap as the term "brocyte" is also used in histopathologic literature as a synonym for "mature" broblasts, Fibrocytes can be obtained and/or differentiated in vitro from the complete peripheral blood mononuclear cell (PBMC) population as well as from an enriched CD14+ population (Abe et al., 2001, Pilling et al., 2009, García-de-Alba et al., 2010). Accordingly, fibrocytes represent one of the variety of cell types that can differentiate from monocytes, including macrophages, osteoclasts and dendritic cells (Wu & Madri 2010; Seta et al., 2010; Castiello et al., 2011).

The fibrocytes obtained from human or mouse blood, either from PBMCs or CD14+ enriched cells, are grown commonly in Dubelcco´s Modified Eagle Medium (DMEM) supplemented with 20% human AB serum (HAB) or fetal calf or bovine serum without the addition of any other growth factors. Some authors have reported the use of RPMI instead of DMEM with good results (Curnow et al., 2010). The resulting fibrocyte population (≥95% pure) is then characterized based on the combined expression of extracellular surface markers including cluster of differentiation (CD) antigens, major histocompatibility complex (MHC)-like molecules, extracellular matrix protein (ECM) markers, and chemokines receptors expression patterns (Metz, 2003) (table 1).


Table 1. Human fibrocytes surface and intracellular phenotype. Reviewed in Bucala et al., 1994, Chesney et al., 1998, Abe et al., 2001, Hartlapp et al., 2001

Hematopoietic Derived Fibrocytes: Emerging Effector Cells in Fibrotic Disorders 321

Flow cytometry is a critical technique for the characterization and quantification of circulating fibrocytes after their enrichment and in vitro differentiation as well as for

**Cell preparation**. Flow analysis requires a single cells suspension. Ice cold 0.05% EDTA in PBS or trypsin-EDTA 0.05% are recommended to detach the cells from the plastic surface, just covering it for 1-2 min at 37° C. Since trypsin is toxic for the cells, they must be observed closely to adjust and change the timing of the trypsin digestion. Immediately, media complemented with 10% serum is added to neutralize the enzymatic activity of the trypsin present in the buffer (normal human AB serum, FCS or FBS). Horizontal shear force can be applied, or cells can be gently scraped if needed for harvesting and they are immediately washed in cold PBS. The number of dead cells should be estimated by trypan

**Number of cells required for staining.** Approximately 2.5-5 x 105 cells with a minimum volume of 300ul of staining buffer (1% BSA-PBS) in polystrene tubes 12X75 are needed for the analysis of the in vitro cultured fibrocytes; fewer cells mean longer collection time and potentially more background noise. For the analysis of circulating fibrocytes from fresh blood samples, ~ 0.5- 1 × 106 cells in 300ul of staining buffer (1% BSA-PBS) in polystrene tubes 12X75 are needed, since the normal percentage of this cells in the circulation is 0.1- 0.5% of the total leukocytes it is better to analyze at least 50,000 events, the use of high

**Protocol for staining cell surface and intracellular antigens for fibrocytes analysis**. The

Cells are centrifuged and resuspended in staining buffer (1% BSA-PBS). The optimum amount of buffer to incubate depends on the protocol suggested by the antibody´s manufacturer technical data sheet, commonly 100μl is an adequate volume. Cells are incubated with the corresponding fluorochrome-labeled antibodies for surface markers (i. e., CD45, CD34, CD11b, and CXCR4) and then fixed and permeabilized with a commercial kit recommended for this purpose (i.e., BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit, BD Biosciences) prior to staining with the anti-collagen antibody or its corresponding isotype control. It is important to consider that isotype controls are critical in the analysis of these cells, since they have to be permeabilized and fixed and a high percentage of nonspecific binding can occur. Also non-stained cells treated with the same process are required as control to discriminate collagen fibers autofluorescence. The number of markers that can be analyzed depend on the capacity (i.e., lasers, filters) of the cytometer to be used, at least 2- 3 fibrocyte markers (i.e., CD45+/Collagen I or CD45+/CXCR4+/Collagen I) are needed to meet the

Fibrocytes increase the expression of α-smooth muscle actin (α-SMA) spontaneously in culture, and gradually loose the expression of CD34 and CD45 over time, which likely reflects terminal differentiation or other phenomena related specifically to a particular tissue microenviroment (Schmidt et al., 2003, Mori et al., 2005, Bucala, 2008). The differentiation of

following steps are the same for both cell types (fresh PBMC´s or cultured cells).

**3.1 Flow cytometry analysis** 

blue exclusion.

fibrocytes obtained directly from fresh blood samples.

performance flow cytometers is recommended.

minimum criteria of the fibrocytes definition.

**3.2 Fibrocyte to myofibroblast differentiation** 

It was previously reported that the differentiation of fibrocytes is inhibited by serum amyloid P (SAP), a major constituent of serum, (Pilling et al., 2003) and more recently it was described that in the absence of serum the process of differentiation of fibrocytes can be accelerated with cells with the spindle-shaped morphology appearing in culture after only ~2-5 days, compared to ~8-14 days when fibrocytes are cultured with serum supplemented medium (Curnow et al., 2010). They also reported a difference in the ability of serum free and serum complemented fibrocytes to differentiate from PBMC and CD14+ peripheral blood cells, with more efficient generation of fibrocytes from PBMC cultured without serum, and from CD14+ cells when these were cultured in the presence of serum complemented medium (Curnow et al., 2010).

Cell population obtained regardless of the initial method for enrichment (PBMC or CD14+ enriched cell culture) are cells expressing a combination of CD45 or other haematopoietic markers (CD34, CD11b), as well as collagen I and III, with an elongated spindle-shaped morphology, making clear that the cells differentiated under both conditions can be classified as fibrocytes, based on the current definition: spindle-shaped cell that expresses both haematopoietic and mesenchymal cell markers, (Bucalla et al., 1994, C. Metz 2003),

Fig. 1. Schematic description of the two most common methods for fibrocytes culture and enrichment.

#### **3.1 Flow cytometry analysis**

320 Advances in Hematopoietic Stem Cell Research

It was previously reported that the differentiation of fibrocytes is inhibited by serum amyloid P (SAP), a major constituent of serum, (Pilling et al., 2003) and more recently it was described that in the absence of serum the process of differentiation of fibrocytes can be accelerated with cells with the spindle-shaped morphology appearing in culture after only ~2-5 days, compared to ~8-14 days when fibrocytes are cultured with serum supplemented medium (Curnow et al., 2010). They also reported a difference in the ability of serum free and serum complemented fibrocytes to differentiate from PBMC and CD14+ peripheral blood cells, with more efficient generation of fibrocytes from PBMC cultured without serum, and from CD14+ cells when these were cultured in the presence of serum complemented

Cell population obtained regardless of the initial method for enrichment (PBMC or CD14+ enriched cell culture) are cells expressing a combination of CD45 or other haematopoietic markers (CD34, CD11b), as well as collagen I and III, with an elongated spindle-shaped morphology, making clear that the cells differentiated under both conditions can be classified as fibrocytes, based on the current definition: spindle-shaped cell that expresses both haematopoietic and mesenchymal cell markers, (Bucalla et al.,

> **Human leukopheresis packs or fresh blood by gradient centrifugation over Ficoll-Paque**

**The final fibrocyte enrichment is routinely ≥95%, as determined by flow cytometry with one of the different combinations of fibrocyte markers (ie, Col I+, CD45+ , CXCR4+)**

Fig. 1. Schematic description of the two most common methods for fibrocytes culture and

**Deplete contaminant leukocyte subsets by positive or negative CD14+ immunomagnetic selection (one step)**

> **Culture CD14+ monocytes in DMEM 20% HAB (FCS or FBS) 8- 10days.**

medium (Curnow et al., 2010).

**Culture resultant PBMCs in DMEM 20% HAB (FCS or FBS) 10-14 days**

**Deplete contaminant leukocyte subsets by immunomagnetic selection using anti-CD3 (T lymphocytes); anti-CD19 (B lymphocytes); and anti-CD14 (monocytes) beads. (3 steps)** 

1994, C. Metz 2003),

enrichment.

Flow cytometry is a critical technique for the characterization and quantification of circulating fibrocytes after their enrichment and in vitro differentiation as well as for fibrocytes obtained directly from fresh blood samples.

**Cell preparation**. Flow analysis requires a single cells suspension. Ice cold 0.05% EDTA in PBS or trypsin-EDTA 0.05% are recommended to detach the cells from the plastic surface, just covering it for 1-2 min at 37° C. Since trypsin is toxic for the cells, they must be observed closely to adjust and change the timing of the trypsin digestion. Immediately, media complemented with 10% serum is added to neutralize the enzymatic activity of the trypsin present in the buffer (normal human AB serum, FCS or FBS). Horizontal shear force can be applied, or cells can be gently scraped if needed for harvesting and they are immediately washed in cold PBS. The number of dead cells should be estimated by trypan blue exclusion.

**Number of cells required for staining.** Approximately 2.5-5 x 105 cells with a minimum volume of 300ul of staining buffer (1% BSA-PBS) in polystrene tubes 12X75 are needed for the analysis of the in vitro cultured fibrocytes; fewer cells mean longer collection time and potentially more background noise. For the analysis of circulating fibrocytes from fresh blood samples, ~ 0.5- 1 × 106 cells in 300ul of staining buffer (1% BSA-PBS) in polystrene tubes 12X75 are needed, since the normal percentage of this cells in the circulation is 0.1- 0.5% of the total leukocytes it is better to analyze at least 50,000 events, the use of high performance flow cytometers is recommended.

**Protocol for staining cell surface and intracellular antigens for fibrocytes analysis**. The following steps are the same for both cell types (fresh PBMC´s or cultured cells).

Cells are centrifuged and resuspended in staining buffer (1% BSA-PBS). The optimum amount of buffer to incubate depends on the protocol suggested by the antibody´s manufacturer technical data sheet, commonly 100μl is an adequate volume. Cells are incubated with the corresponding fluorochrome-labeled antibodies for surface markers (i. e., CD45, CD34, CD11b, and CXCR4) and then fixed and permeabilized with a commercial kit recommended for this purpose (i.e., BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit, BD Biosciences) prior to staining with the anti-collagen antibody or its corresponding isotype control. It is important to consider that isotype controls are critical in the analysis of these cells, since they have to be permeabilized and fixed and a high percentage of nonspecific binding can occur. Also non-stained cells treated with the same process are required as control to discriminate collagen fibers autofluorescence. The number of markers that can be analyzed depend on the capacity (i.e., lasers, filters) of the cytometer to be used, at least 2- 3 fibrocyte markers (i.e., CD45+/Collagen I or CD45+/CXCR4+/Collagen I) are needed to meet the minimum criteria of the fibrocytes definition.

#### **3.2 Fibrocyte to myofibroblast differentiation**

Fibrocytes increase the expression of α-smooth muscle actin (α-SMA) spontaneously in culture, and gradually loose the expression of CD34 and CD45 over time, which likely reflects terminal differentiation or other phenomena related specifically to a particular tissue microenviroment (Schmidt et al., 2003, Mori et al., 2005, Bucala, 2008). The differentiation of

Hematopoietic Derived Fibrocytes: Emerging Effector Cells in Fibrotic Disorders 323

recently been reported that fibrocytes possess the ability to differentiate into chondrocytes and osteoblasts in vitro when the appropriate combination of cytokines and growth factors are used (Choi et al., 2010). These ndings, taken together with their capacity to differentiate into myofibroblasts and adipocytes, indicate that fibrocyte may differentiate toward several types of mesenchymal cell types and that this process is inuenced by a complex prole of

**Induction of the differentiation of fibrocytes to osteoblasts:** Puried brocytes are seeded at a concentration of 1×105 cells/well in a bronectin-coated 12 well plate, they are treated with osteogenic basal media (this media is commercially available) supplemented with dexamethasone, ascorbate, mesenchymal cell growth supplement (MCGS), l-glutamine, 1× Penicillin/Streptomycin, and β-glycerophosphate. β-glycerophosphate is critical to stimulate calcied matrix formation in combination with the effects of dexamethasone and ascorbate. Cells are cultured during 21 days with media replacement every 3 days (Choi et

**Induction of the differentiation of fibrocytes to chondrocytes:** Puried brocytes are seeded at the concentration of 5×104 cells/tube in 15ml sterile polypropylene tubes, followed by centrifugation at ∼300×g for 10min to form pellets. Supernatant has to be carefully removed in order not to disrupt the brocyte micromass pellet. Fibrocytes are additioned with chondrogenic differentiation cocktail: basal chondrogenic media (also commercially available) supplemented with 1 x 10-7M dexamethasone, 0.1 M ascorbate, lglutamine, Penicillin/Streptomycin, 1 M sodium pyruvate, proline and 10 ng/ml of TGF-β3. Cells are cultured for 21 days with media replacement every 2 - 3 days (Barry et al., 2001;

Wound repair is a complex process that results from the coordinated release of cytokines, chemokines, and growth factors, leading successively to the recruitment and activation of different cells into the injured site from the very initial phases of repair (Gurtner GC et al., 2008). Fibrocytes have been postulated as important players of the tissue repair process since they have the ability to rapidly home to sites of tissue together with the infiltrating inflammatory cells that act to prevent infection and degrade damaged connective tissue

Fibrocytes secrete proinammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin (IL)-6, IL-8, IL-10, macrophage inammatory protein-1α/β (MIP-1α/β) CCchemokine ligands (CCL) -3 and -4 in response to IL-1β which is an important mediator of wound healing response (Chesney et al., 1998). The fibrocyte products MIP-1α , MIP-1β, and monocyte chemotactic protein-1 (MCP-1) are potent T cell chemoattractants and may act to specifically recruit CD4+ T cells into the tissue repair microenvironment; moreover, the fibrocytes increase the cell surface expression of leukocyte adhesion molecules, such as intercellular adhesion molecule 1 (ICAM1), which would enhance leukocyte trafficking (Chesney et al., 1998). Interestingly, in addition to these functions, fibrocytes may play an early and important role in the initiation of antigen-specific immunity. Thus, it has been demonstrated that peripheral blood fibrocytes: express the surface proteins required for antigen presentation, including class II major histocompatability complex molecules: HLA-

cytokines within the local microenvironment of the host tissue or tissue injury.

al., 2010).

Choi et al., 2010). Fig. 2.

components (Bucala et al., 1994).

**4. Fibrocytes participation in repair processes** 

brocytes into myobroblasts can be enhanced by transforming growth factor (TGF)-β or endothelin-1, which results in an increment in the synthesis of collagen and the myobroblast marker α–SMA (Schmidt et al., 2003; Bucala, 2008).

**For myofibroblast differentiation as described in the literature (Hong et al., 2007):** a population of enriched fibrocytes has to be previously obtained with one of the techniques described above. The percentage of enrichment needs to be verified by flow cytometry in each culture to ensure reproducibility of the results.

Fibrocytes are treated with serum-free DMEM with 10 ng/ml TGF-β1 for 3 weeks, refreshing TGF-β1 supplemented medium every 48-72hs. If the objective is to analyze changes in the pattern of gene and protein expression, time curves should be performed previously since these effects might be different depending on the gene or protein of interest.

The signaling pathways that are activated by TGF-β1 to induce α-SMA transcription and thus brocyte differentiation to myobroblast-like cells include Smad2/3 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) - mitogen-activated protein kinase (MAPK). Interestingly, it was reported that treatment with troglitazone (TGZ, a synthetic agonist of peroxisome proliferator-activated receptor gamma: PPARγ), inhibits TGF-β1 induced α-SMA expression and this effect is modulated through attenuation of the SAPK/JNK activity leading to decreased Smad2/3 levels and transactivation activity, (Hong et al., 2007).

#### **3.3 Adipocyte differentiation**

Hong et al., demonstrated in a model of differentiation of human circulating adipogenic progenitors to adipocytes in SCID mice, that brocytes, in the presence of specic environmental characteristics can give rise to adipocytes. By gene microarray analysis they found a signicant up-regulation of specic mature adipocyte genes and proteins after brocyte differentiation to adipocyte, including fatty acid binding protein 4 (FABP4), leptin, and PPARγ; remarkably certain genes, such as those involved in cell motility, chemotaxis, or metalloproteinase activity where also upregulated in the process of differentiation to adipocytes. These findings indicate that fibrocytes may retain unique functions for motility and chemoattractive activity that might allow them to participate in migration and trafcking despite their differentiation into adipocytes (Hong et al., 2005). Differentiation of fibrocytes into adipocytes appears to be mediated by PPARγ that leads to lipid accumulation and induction of aP2 gene expression (Rival et al., 2004). By contrast, this process is inhibited by TGF-β through SAPK/JNK pathway activation (Hong et al., 2007).

**For adipocyte differentiation**: fibrocytes are treated with PBM culture media (Cambrex Bio Science) supplemented with 10 M troglitazone. Culture media has to be changed every 48 h for 21 days. Following 21 days in culture, the cells accumulate lipids in intracellular vacuoles. Oil Red O Staining can be used to confirm fibrocytes differentiation to adipocytes (Hong et al., 2007).

#### **3.4 Osteoblast and chondrocyte differentiation**

Osteoblasts and chondrocytes, which are derived from a common mesenchymal precursor cell, are critical in bone and cartilage formation respectively (Knothe et al., 2010). It has

brocytes into myobroblasts can be enhanced by transforming growth factor (TGF)-β or endothelin-1, which results in an increment in the synthesis of collagen and the

**For myofibroblast differentiation as described in the literature (Hong et al., 2007):** a population of enriched fibrocytes has to be previously obtained with one of the techniques described above. The percentage of enrichment needs to be verified by flow cytometry in

Fibrocytes are treated with serum-free DMEM with 10 ng/ml TGF-β1 for 3 weeks, refreshing TGF-β1 supplemented medium every 48-72hs. If the objective is to analyze changes in the pattern of gene and protein expression, time curves should be performed previously since these effects might be different depending on the gene or protein of

The signaling pathways that are activated by TGF-β1 to induce α-SMA transcription and thus brocyte differentiation to myobroblast-like cells include Smad2/3 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) - mitogen-activated protein kinase (MAPK). Interestingly, it was reported that treatment with troglitazone (TGZ, a synthetic agonist of peroxisome proliferator-activated receptor gamma: PPARγ), inhibits TGF-β1 induced α-SMA expression and this effect is modulated through attenuation of the SAPK/JNK activity leading to decreased Smad2/3 levels and transactivation activity, (Hong et al., 2007).

Hong et al., demonstrated in a model of differentiation of human circulating adipogenic progenitors to adipocytes in SCID mice, that brocytes, in the presence of specic environmental characteristics can give rise to adipocytes. By gene microarray analysis they found a signicant up-regulation of specic mature adipocyte genes and proteins after brocyte differentiation to adipocyte, including fatty acid binding protein 4 (FABP4), leptin, and PPARγ; remarkably certain genes, such as those involved in cell motility, chemotaxis, or metalloproteinase activity where also upregulated in the process of differentiation to adipocytes. These findings indicate that fibrocytes may retain unique functions for motility and chemoattractive activity that might allow them to participate in migration and trafcking despite their differentiation into adipocytes (Hong et al., 2005). Differentiation of fibrocytes into adipocytes appears to be mediated by PPARγ that leads to lipid accumulation and induction of aP2 gene expression (Rival et al., 2004). By contrast, this process is inhibited by TGF-β through SAPK/JNK pathway activation (Hong et al., 2007).

**For adipocyte differentiation**: fibrocytes are treated with PBM culture media (Cambrex Bio Science) supplemented with 10 M troglitazone. Culture media has to be changed every 48 h for 21 days. Following 21 days in culture, the cells accumulate lipids in intracellular vacuoles. Oil Red O Staining can be used to confirm fibrocytes differentiation to adipocytes

Osteoblasts and chondrocytes, which are derived from a common mesenchymal precursor cell, are critical in bone and cartilage formation respectively (Knothe et al., 2010). It has

myobroblast marker α–SMA (Schmidt et al., 2003; Bucala, 2008).

each culture to ensure reproducibility of the results.

interest.

**3.3 Adipocyte differentiation** 

(Hong et al., 2007).

**3.4 Osteoblast and chondrocyte differentiation** 

recently been reported that fibrocytes possess the ability to differentiate into chondrocytes and osteoblasts in vitro when the appropriate combination of cytokines and growth factors are used (Choi et al., 2010). These ndings, taken together with their capacity to differentiate into myofibroblasts and adipocytes, indicate that fibrocyte may differentiate toward several types of mesenchymal cell types and that this process is inuenced by a complex prole of cytokines within the local microenvironment of the host tissue or tissue injury.

**Induction of the differentiation of fibrocytes to osteoblasts:** Puried brocytes are seeded at a concentration of 1×105 cells/well in a bronectin-coated 12 well plate, they are treated with osteogenic basal media (this media is commercially available) supplemented with dexamethasone, ascorbate, mesenchymal cell growth supplement (MCGS), l-glutamine, 1× Penicillin/Streptomycin, and β-glycerophosphate. β-glycerophosphate is critical to stimulate calcied matrix formation in combination with the effects of dexamethasone and ascorbate. Cells are cultured during 21 days with media replacement every 3 days (Choi et al., 2010).

**Induction of the differentiation of fibrocytes to chondrocytes:** Puried brocytes are seeded at the concentration of 5×104 cells/tube in 15ml sterile polypropylene tubes, followed by centrifugation at ∼300×g for 10min to form pellets. Supernatant has to be carefully removed in order not to disrupt the brocyte micromass pellet. Fibrocytes are additioned with chondrogenic differentiation cocktail: basal chondrogenic media (also commercially available) supplemented with 1 x 10-7M dexamethasone, 0.1 M ascorbate, lglutamine, Penicillin/Streptomycin, 1 M sodium pyruvate, proline and 10 ng/ml of TGF-β3. Cells are cultured for 21 days with media replacement every 2 - 3 days (Barry et al., 2001; Choi et al., 2010). Fig. 2.
