The effects of BMP-4, FGF-2, and PTH using a micromass culture technique to examine

chondrogenesis in chick limb buds

What is Chondrogenesis?

Chondrogenesis is the earliest phase of skeletal development, which involves:

1. mesenchymal cell recruitment and migration

2. condensation of progenitors

3. chondrocyte differention and maturation

4. results in the formation of cartilage and bone during endochondral ossification

How does chondrogenesis occur?

Chondrogenesis is initiated by the differentiation of mesenchymal cells thatarise from three sources:

1. neural space cells of the neural ectoderm that gives rise to craniofacial bones

2. the sclerotome of the paraxial mesoderm which forms the axial skeleton

3. the somatopleure of the lateral plate mesoderm which yields the skeleton of the limbs

The sequential events of chondrogenesis

These sequential events are dependent on the following:

1. cell-cell interactions

2. cell-extracellular matrix interactions

3. autocrine/paracrine growth factor regulations

Endochondral and intramembranous bone

development

Diagram of endochondral ossification

1. Mesenchymal cells commit to becoming cartilage cells (chondrocytes)

2. Committed mesenchyme condenses into compact nodules

3. Nodules differentiate into chondrocytes and proliferate to form the cartilage model of bone

4. Chondrocytes undergo hypertrophy and apoptosis while they change and mineralize their extracellular matrix

5. Apoptosis of chondrocytes allows blood vessels to enter

6. Blood vessels bring in osteoblasts which bind to degenerating cartilaginous matrix and deposit bone matrix

7. Bone formation and growth consists of ordered arrays of proliferating, hypertrophic, and mineralizing chondrocytes

The stages of endochondral ossification

Some growth factors that promote chondrogenesis

BMPs (Bone Morphogenetic Proteins)

FGFs (Fibroblast Growth Factors)

PTH (Parathyroid Hormone)

What are Bone Morphogenetic Proteins?

Bone Morphogenetic Protein comes from the family of secreted growth factors which forms a subgroup of molecules within the transforming

growth factor β (TGF-β) superfamily.

What does BMP do and how does it promote chondrogenesis?

BMP promotes ectopic cartilage and bone formation.

Other roles include regulation of:

cell division

apoptosis

cell migration

differentiation

What are FGFs?

FGFs are a family of polypeptides that have important roles in:

★cell growth

★differentiation

★survival

★many other developmental processes

What is FGF’s role in chondrogenesis?

✤ is a strong modulator of the maturation process of chondrocytes during endochondral ossification

✤ has also been used in many progenitor cell culture systems to stimulate proliferation.

✤ plays an important role in maintaining self renewal capacities of embryonic and adult progenitor cells.

✤ has beneficial effects on chondrogenesis and cartilage defects

What does parathyroid hormone (PTH) do?

Parathyroid hormone acts in a similar fashion as FGF-2 by stimulating the maturation process of:

✦ chondrocytes

✦cell proliferation

✦matrix synthesis

PTH plays an important role in regulating the growth and metabolism of the developing skeletal elements (cartilage and bone)

FGF in developing Limb

My experiment

I looked at chondrogenesis in chick limb bud cells in vitro by testing three drugs:

1. Parathyroid hormone (PTH)(0.25 ul of 2x10-7 M)

2. FGF-2 (10 ng/ml)

3. BMP-4 (10 ug/2 ml)

My control consisted of complete medium with serum and antibiotic.

✴nodule formation

Materials and MethodsFive 4-day old chick embryos were collected and placed into a sterile petri dish containing sterile Howard’s Ringers.

By using forceps, all four of the limb buds were removed from each embryo and placed into a sterile 15 ml plastic centrifuge tube (with 1 ml of 0.1% trypsin at 37 degrees Celsius.)

Ten minutes later, this liquid was removed and replaced with 2 ml of complete medium (medium + serum, antibiotics, and glutamine) before triturating to dissociate the cells.

Materials and Methods (continued...)

Pieces of the chick limb buds were filtered using an autoclaved Swinnex (containing a nylon mesh screen), which was attached to a 3 ml syringe barrel.

The cells were counted using a hemocytometer.

The cells were centrifuged for 3 minutes on setting 5 using the clinical centrifuge.

By leaving the cell pellet undisturbed, the medium was removed and discarded by using a sterile Pasteur pipet.

Materials and Methods continued. . .

Enough fresh complete medium was added to make a cell concentration of 30 million cells/ml.

Three spots of cells at concentrations of 30, 15, and 5 million cells/ml were evenly spaced in the formation of an imaginary triangle in 35 mm tissue culture plates.

Materials and Methods continued...

❖ The cells were allowed to attach to the covered culture plates for 30 minutes at room temperature.

❖ The three drugs with the following concentrations were tested:

1. Parathyroid hormone (0.25 ul of 2 x 10 7 M)

2. FGF-2 (10 ng/ml)

3. BMP-4 (10 ug/2 ml)

3. Control (complete medium with serum and antibiotic)

❖ The culture plates were then placed into the 5% carbon dioxide gas flow incubator at 37 degrees Celsius for one and three days.

Incubation and Fixing with Formaldehyde

• I also looked at the effect of time on cartilage nodule formation by taking 2 of each condition (PTH, BMP-4, FGF-2, and control) out of the carbon dioxide incubator after 1 day, 2 days, 4 days, and 5 days.

• The medium was then:

• 1. removed from the culture

• 2. rinsed twice with Howard’s Ringers

• 3. covered with phosphate buffered 10% formalin

• 4. stored in the refrigerator

Hypothesis: I thought that the samples that were incubated for a longer period of time (before fixing them with formaldehyde)would have more cartilage nodules. I believed that BMP-4 would have the most nodules because it causes bone formation.

Characterizing cartilage nodules

The aggregation phase is characterized by the expression of cartilage-specific proteoglycan, chondroitin sulfate.

I used a phase microscope to identify the cartilage nodules.

How are the nodules characterized?

The nodules can be seen by using toluidine blue dye, which complexes with acidic polymers like chondroitin sulfate.

Staining and scoring the nodules

• The formalin was removed and rinsed with distilled water

• The cell layer was covered with 1% toluidine blue for 5 minutes.

• This was rinsed twice with water and twice with ethanol.

• The cell layer was covered with one ml of PBS. The number of distinct nodules were scored.

Results

Control Day 2

BMP4 Day 2

FGF2 Day 2

PTH Day 2

Control Day 4

BMP4 Day 4

FGF2 Day 4

PTH Day 4

FGF and BMP signaling pathways

BMP action in chondrogenesis

Parathyroid hormone signaling

Other experiments looking at the effects of

BMP, FGF, and PTH on chondrogenesis

BMP-4 research

✴Local delivery of BMP-4 by genetically engineered muscle-derived stem cells enhanced chondrogenesis and significantly improved articular cartilage repair in rats

✴BMP is involved in both cartilage repair by periosteal chondrogenesis and fracture repair

✴BMP initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP an excellent regulator of skeletal cell differentiation

✴Dermal fibroblast-mediated BMP-2 therapy accelerates bone healing in equine metacarpal/metatarsal osteotomies.

Research showing the effects of FGF-2 on chondrogenesis

FGF-2 used to treat bone marrow-derived mesenchymal stem cell monolayers enhanced subsequent chondrogenic differentiation in a 3-D culture.

It is found to be up-regulated early in condensing mesenchyme.

Investigations showing the effects of parathyroid hormone on

chondrogenesis✓ Parathyroid hormone and FGF-2 are

negative regulators of chondroctye maturation, and they are required for normal skeletal development. If these regulators are absent or defective, most, if not all, chondrocytes can still undergo maturation and be replaced by bone cells.

✓ PTH is responsible at the onset of chondrogenesis and osteogenesis in vivo within embryonic chick limb buds.

Literature Cited

• Ballard, T. A. and Biddulph, D.M. (1984). The morphology and hormonal responsiveness of developing skeletal elements in chick limb buds. Am. J. Anat. 169, 221-236.

• Goldring, M. B., Tsuchimochi, K., and Ijiri, K. (2006). The control of chondrogenesis. J. Cell. Biol. 97, 33-44.

• Ishihara, A., Zekas, L. J., Litsky, A. S., Weisbrode, S. E., and Bertone, A. L. (2009). Dermal fibroblast-mediated BMP-2 therapy to accelerate bone healing in an equine osteotomy model. J. Orthop. Res. 28, 403-411.

• Iwamoto, M., Shimazu, A., and Pacifici, M. (1995). Regulation of chondrocyte maturation by fibroblast growth factor-2 and parathyroid hormone. J. Orthop. Res. 13, 838-845.

• Liu, Z., Xu, J., Colvin, J. S., and Ornitz, D. M. (2002). Coordination of chondrogenesis and osteogenesis y fibroblast growth factor 18. Genes & /Dev.

Literature Cited

• Liu, Z., Xu, J., Colvin, J. S., and Ornitz, D. M. (2002). Coordination of chondrogenesis and osteogenesis y fibroblast growth factor 18. Genes & /Dev. 16, 859-869.

• Loveys, L. S., Gelb, D., Hurwitz, S. R., Puzas, J. E., and Rosier, R.N. (1993). Effects of parathyroid hormone-related peptide on chick growth plate chondrocytes. J. Orthop. Res. 11, 884-891.

• Nonaka, K., Shum, L., Takahashi, I., Takahashi, K., Ikura, T., Dashner, R., Nuckolls, G. H., and Slavkin, H. C. (1999). Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis. Int. J. Dev. Biol. 43, 795-807.

• Kuroda, R., Usas, A., Kubo, S., Corsi, K., Peng, H., Rose, T., Cummins, J., Fu, F. H., Huard, J. (2006). Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells. Arthritis. Rheum. 54, 433-442.

Literature Cited

• Rosen, V., Nove, J., Song, J. J., Thies, R. S., Cox, K., and Wozney, J. M. (1994). Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes. J. Bone Miner. Res. 9, 1759-1768.

• Sanyal, A., Sarkar, G., Saris, D. B. F., Fitzsimmons, J. S., Bolander, M. E., and O’Driscoll, S. W. (1999). Initial evidence for the involvement of bone morphogenetic protein-2 early during periosteal chondrogenesis. J. Orthop. Res. 17, 926-934.

• Stewart, A. A., Byron, C. R., Pondenis, H., and Stewart, M. C. (2007). Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. Am. J. Vet. Res. 68, (9), 941-945.

• Yoon, B. S. and Lyons, K. M. (2004). Multiple functions of BMP’s in chondrogenesis. J. Cell. Biochem. 93, 93-103.