Glycosaminoglycans:

This family of carbohydrates is essential or important for life.

GAGs form an important component of connective tissues.

is a stiff yet flexible connective tissue found in many areas in the bodies of humans and other animals

Cartilage is composed of specialized cells called chondrocytes that produce a large amount of extracellular matrix composed of collagen fibers, abundant ground substance rich in proteoglycan, and elastin fibers. Cartilage is classified in three types, elastic cartilage, hyaline cartilage and fibrocartilage, which differ in the relative amounts of these three main components

Unlike other connective tissues, cartilage does not contain blood vessels. The chondrocytes are supplied by diffusion, helped by the pumping action generated by compression of the articular cartilage or flexion of the elastic cartilage

They have a core protein with one or more covalently attached glycosaminoglycan (GAG) chain(s)Proteoglycans occur in the connective tissue.

Proteoglycans are a major component of the animal extracellular matrix, the "filler" substance existing between cells in an organism. Here they form large complexes, both to other proteoglycans, to hyaluronan and to fibrous matrix proteins (such as collagen). They are also involved in binding cations (such as sodium, potassium and calcium) and water, and also regulating the movement of molecules through the matrix. Evidence also shows they can affect the activity and stability of proteins and signalling molecules within the matrix. Individual functions of proteoglycans can be attributed to either the protein core or the attached GAG chain.

k = Qh/AΔP

k: hydraulic permeability m4/N•s
Q: flow rate m3/s
h: specimen thickness
A: cross-sectional area m2
ΔP: P1 – P2 N/m2

used to resorb bone, induces bone resorption, stimulated when you have low calcium levels in the blood

affects kidney permeability for ions (reduces the ammount of phosphorous)

Induces bone formation,

stimulated by too much calcium in the blood serum

If in low gravity, reduces Ca levels in body

Skullcap,

has suture lines (fibroblasts) to allow skull to grow

If lines are fused, causes serious damage

Mem + 10% FBS

Floating, looks at response to different hormones

Mechanosensing cell

Lacuna-> has osteocyte in the inside

Sensor cells

multinucleated cells which resorb bone

hematopoetic cells, sit around and spit out acid (looks like packman)

acid phosphatase

alkaline phosphatase is released and can possibly form bone depending, responsible for changing phosphate saturation levels

Collagenase/trypsin (Ca2+) digestion

Filter thoguh digestion system (filter paper depends on cells)

spin down & add more media

Repeat this 5 times to get more pure cells

1-2: Perifibroblasts, fibroblasakosteoclasts
3: osteoblast like -> day 5

increase surface areas for diffusion

creates a physiological 3D environment

Acts as a sieve to trap products

PGA-> Polyglycolic acid (fibrous scaffold), typically 100-200nm distance between fibresr, 10-15um in diameter, breaks down in culture and situ through hydrolosis, used for sutures

Poly-L-Lactic acid (PLLA)- moy hydrophobic than PGA, dissolved in methlene chloride

derived from Kelp, polysaccharide (thickening agent for food)

polymerization in duced by divalent ions, Ca2+, Mg2+ (CaCl2, CaSO4)

Alganate powder + media + cells, add CaCl2 -> gels

inject into defect, in situ polymerization

Degrades in culture -> Na2+ to NaCl2 in media

swaps out Ca2+

Ba2+ and Sr2+ irreversibly crosslinks

breaks down Ca2+ Crosslinks

extract cells for gene expression

the cell matrix can be use ascaffold system

Attachment or encapsulation, polymers that permit protien adsorbtion will allow direct cell attachment

Traps cells in hydrogel like alginate agorose-> hydrophilic - > discourages protien adsorption

70% ethyl alcohol

autoclave

ethlene oxide gas

irradiation

sterile filther (0.22um)

Wells must be deep enough to allow for cells not growing out
This creats a difference in media as cells in wells act differentl

there's is a concentration gradient in the media but if you stir it up it changes

when media comes into a well cells try getting out

gelling via CaCl2 -> minutes, CaSO4 -> 30-40min @4C

Disk w/ cells + alginate, filter paper, CaCl on top (concentration physiologic osmolarity ~100mM in 100mosm solution)

Cells are in liquid , pour the CaCl into alginate

CaCl does not pass through alginate as it's too viscous (add dextran 2Mmolecules weight) add stir bar to allow for mixture

Calcium begins to leave and once there is no more Ca inside it will stop propogating. Depends on the conentration of Ca

subcutaneous model, under the skin implanted, nutrients from surrounding tissue

(controls)

cells + scaffold

scaffold alone

cells alone

nothing

Native cartilage breaks down in situ fast

engineered tissues grow (PGA + cells) depends on the cell density in tissue

Phase 1: plating expansion (Fibroblasts derived growth factor)

Phase 2: cell seeding

Phase 3: 3D culture

Progenitor cells-> long bone

flush marrow adhesion for cell selection (stromal)

Source: iliac crest (hip bone), neck biopsy.> autologous cell source-> progenitor-> can become different cell types -> tissues

Looking how to push to desired phenotype

Cell seeding in PGA constructus

use a stirbar to create flow in jar, where four constructus are hanging inside waiting for cells to get inside constructus

Effeciency of seeding can be determined how many cells you add and cells in tissue.

Cuture phase in 3D

orbital shaker, layer agarose

Find the effect on tissue growth growth factors makign tissue (cells living in 3D)

Cartilage, bone, control pellet culture (no scaffold)

Add dexamethasone

B-glycerophosphate

(causes alkaline phosphate, type I collagen, osteocalcin, mineralization)

Inhomogenious, cells change size

Growth plate cartilage -> type X collagen

Holds strength, cells form tissue and stiffness generally degrades as cells make more scaffold

from pellet takes longer to make tissue but eventually forms it

Alginate can pull cells out with

Agarose, cannot pull cells out, irreversable crosslinked

PEG can break down over time (respond from collagnase, cells make collaganase, and then breakdown PEG)

cells attach on outside and form "pseudo 2d" and make collage type 1 while inside make collegen type 2

Cells on outside are strong in tension, whcih holds cells on inside intact, but if punched, loose all strength

if you use too large concentration, pores too small (weight/volume)

As matrix is made, pores become clogged with matrix

choose a low crosslinking density to allow for cells to grow

Crosslink w/ UV light

Cells are attracted to an attractant

Place cells into well, and seperate it from a growth factor gel (seperated by hydrogel, cause a diffusion gradient across hydroge)

cells in well are in maximum concentration (#cells in well are infinite, as they move they leave them)

Cells wiggle around w/out a gradient

w/ growth factor, cells move around in a larger motion (still no gradient) -> Chemokinesis, random motility of cell is affected by some attractant

Random motility coefficient u(a)(distance²/time

a = attractant

Chemotaxis

x = chemotaxis coefficient (distance²/time)/(moles/unit volume)

shows the bias in cells towards a certain direction

haptotaxis

cells like the surface and move towards it

random motility + chemokinesis + chemotaxis

Gradient diffustion-Gradient in attractiveness

Concentration gradient of cells

C(x,t) (space and time)

dJ/dx = dC/dt =dc(x,t)/dt = dJ(x,t)/dx

dc/dt = ud²c/dx²

u is analogous to "d" in diffusion coeff

chemokinetic

J_c= -udc/dx + (-0.5du(a)/da + x) * cΔa (connective da/dx)

u = 0.5P(s²)

P = persistance time (duration it moves and stops)
s = speed of cell

V_C = Χdel(a) - Xda/dx

= ΦS

where S = speed

Φ = average orientated bias

value = 0  completely directed movement (purely random movement = 1)

Φ = 2f-1

f = 1/2 -> Φ = 0

fraction of cell orientation in a particular direction

*c(x,t) = c_o(1-erf(x/2(ut)^0.5)*

erf(x) = 2/√π int(0-x)e^-t2dt

eft(0) = 0

erf(∞) = 1

erf(-x) = -erf(x)

erf(x) + erfc(x)(complimentary error function) = 1

erf(x) = 1-erfc(x) -> erf^-1(1-x) = erfc^-1(x)

c/c_o - 1-erf (x/2(ut)^0.5

efr^-1(1-c/c_o) = x/2(ut)^0.5

erfc^-1(c/c_o) = x/2(ut)^0.5 = x/(4ut)^0.5

experiment: c_{o = initial concenc}

(x,t) = cell concentration

m = 4(ut)^-0.5

u = (4t²m²)^-1

graphed of erfc over x

Keeps nutrients moving around as cells don't sink since whole thing is moving, boyancy keeps floating

Creates time averaged microgravity

Clintorotation- has problem growing

Slowly drifts from swall

Bone minerals forms around tissue more when you spin