Conference on bone VIBS 443 and VIBS 602

FUNCTIONS OF CARTILAGE EVOLUTIONARY EMRYONIC MODEL FOR BONES

CELLS OF CT FIBROBLASTS MESENCHYMAL CELLS and RBC ADIPOSE CELLS MACROPHAGE PLASMA CELLS MAST CELLS and WBC CHONDROBLASTS CHONDROCYTES OSTEOBLASTS OSTEOCYTES OSTEOCLASTS

HYALINE CARTILAGE EPIPHYSEAL PLATE, RESPONSIBLE FOR LONGITUDINAL GROWTH OF LONG BONES

ENDOCHONDRAL (SPONGY/ CANCELLOUS) BONE FORMATION CARTILAGE MODEL CENTERS OF OSSIFICATION PRIMARY CENTER OF OSSIFICATION • DIAPHYSIS SECOND CENTER OF OSSIFICATION • CENTER OF EACH EPIPHYSIS

MITOSIS DEFINED - CELL DIVISION IN WHICH DUPLICATED CHROMOSOMES DIVIDE INTO DAUGHTER CELLS THAT HAVE IDENTICAL CHROMATIN CONTENT

Developing ovarian follicle has many cells in mitosis

Radioactive precursors is the source of radioactivity in final cellular product --------------------- Product Precursor Protein (general) DNA amino acids thymidine RNA uridine Collagen (sp. protein) proline and lysine (sp. a. a. ) Cartilage matrix 35 SO 4

Tritiated Thymidine labeling of nuclei

DO THE : PULSE – CHASE EXPERIMENT LOOK FOR THE : TEMPORAL APPEARANCE OF SILVER GRAINS IN THE PHOTOGRAPHIC EMULSION OVER ORGANELLES OF INTEREST

EVIDENCE FOR PROTEIN PATHWAY TEMPORAL APPEARANCE OF RADIOACTIVE PROTEINS IN DIFFERENT ORGANELLES (produced from radioactive precursors e. g. , labeled AA) Detected by: AUTORADIOGRAPHY VISUAL CELL FRACTIONATION BIOCHEMICAL

EVIDENCE FOR PROTEIN PATHWAY AUTORADIOGRAPHY PROCEDURE TO LOCALIZE A PRODUCT (e. g. , PROTEIN) WITHIN A CELL OR GEL THAT IS SELF-RADIOACTIVE DUE TO THE CELL’S INCORPORATION OF RADIOACTIVE PRECURSORS (e. g. , RADIOACTIVE AMINO ACIDS) INTO THAT PRODUCT THAT IS VISUALIZED IN A PHOTOGRAPHIC EMULSION.

GENERAL ORGANIZATION OF CARTILAGE CHONDROCYTES / CHONDROBLASTS

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ANGIOGENESIS

ANGIOGENESIS – growth of blood vessels

MESENCHYMAL CELLS Endothelial cells Smooth muscle cells Fibroblasts

COMPACT BONE REMODELING – OSTEOCLASTS DIGEST BONE, OSTEOBLASTS FROM ENDOSTEUM REPLACES IT

CELLS OF BONE OSTEOBLAST OSTEOCYTE OSTEOCLAST

BONE REPAIR

Expected events with bone formation • • • Clot Migration if mesenchymal cells Proliferation Differentiation Calcification Entry of blood vessels, osteoclasts, and osteoblasts • Colonization of bone marrow

Cellular/tissue processes involved • • • Chemotaxis Mitosis Cell differentiation Cell death Angiogenesis

Considerations • How to set up an experiment to test the temporal changes in transplant growth and development? • How to set up an assay for each event in bone formation to evaluate the temporal changes expected in transplants placed in the back skin of rats? • What can be concluded about the cellular and tissue processes involved in the general transplant or transplants receiving specific extracted components?

CONFERENCE ON BONE FORMATION • Because your brother was born with a severe facial disfigurement, you decided to become a plastic surgeon. You dreamed of developing a magic powder that you could mix and form into a mode, that when implanted would stimulate the formation of bone of exactly the right shape and structure that you needed for your reconstructive surgery. Being more than a dreamer, you decided to study bone formation and figure out how to transform your dream into reality. • Just for kicks, you demineralized some rat bone fragments (treat with 0. 5 M HCl, then extract with distilled water, ethanol & ether) to kill all the endogenous cells (such that new bone growth would result from stimulation of host cells and not proliferation of cells from the implant itself) and extract most of the calcium and lipids, and then implanted these fragments under the skin of another rat. Subcutaneous implantation of demineralized bone matrix in rats was found to induce factors in the host resulting in the biosynthesis and differentiation of cartilage, bone, and hematopoietic marrow at the site of the implant. • 1. Design an experiment to test the sequence of events and the time course required for new bone formation. • A. Describe the expected morphology of each stage of new bone formation occurring at the site of the implant.

• B. If you injected radioactive thymidine (cell division) or sulfate (metabolic differentiation) at the site of the implant of developing bone and then made sections of the implanted material followed by autoradiography, which cells and/or structures of the developing bone would have incorporate either label? • 2. In order to try to isolate and characterize some of these factors, you extract the demineralized bone with high salt and SDS, conditions which should extract many proteins. You then implant the bone residue to see if it still promotes bone formation. What stages of bone formation might be blocked, and how could you test where it is blocked? If you hypothesize that your extraction procedure has removed a chemotactic factor necessary for recruitment of mesenchymal cells toward the site of the implant, what tests could you perform to prove your hypothesis? • 3. Your brother's bone disease is postulated to have been due to an overproduction of osteoclasts. Using bone fragments and marrow obtained by a biopsy, what morphological characteristics would you expect his adult bone to have? What results might occur if you implanted demineralized bone fragments from the biopsy into rats? How could you explain your result if the bone implant from the biopsy material resulted in normal bone formation? What tests might you perform if you suspect that the matrix of your brother's bone is the problem?

HISTOGENESIS OF BONE ENDOCHONDRAL OSSIFICATION DEPOSITION OF BONE MATRIX ON A PREEXISTING CARTILAGE MATRIX CHARACTERISTIC OF LONG BONE FORMATION

ENDOCHONDRAL BONE GROWTH IN WIDTH - EXTENSION OF COMPACT BONE BY APPOSITIONAL GROWTH IN LENGTH - EPIPHYSEAL PLATE CARTILAGE GROWTH CALCIFICATION OF CARTILAGE SPONGIOSA • PRIMARY SPONGIOSA – CARTILAGE CORE • SECONDARY SPONGIOSA OSTEOID CORE