Histological Techniques & Analysis

Question 1

What are biomaterials ?

Answer 1

• Exogenous materials which restore function to damaged tissue
• vascular graft, stent, pacemaker

Question 2

What are some issues of biomaterials ?

Answer 2

• biocompatibility
• surface chemistry & topography
• materials escape from graft

Question 3

Define Biocompatibility

Answer 3

• the ability of a biomaterial to perform its function, without eliciting any undesirable local or systemic effects

Question 4

What are the main cells involved in dictating the biocompatibility of implanted material ?

Answer 4

neutrophils, macrophages, lymphocytes, fibroblasts, mast cells, tissue specific cells

Question 5

Describe the role of neutrophils in biocompatibility

Answer 5

• 1st line of immunological defence
• produce wide range of molecules that can damage implants
• key for cell signalling that coordinates tissue healing

Question 6

Describe the role of macrophages in biocompatibility

Answer 6

• clear debris from implant site
• secretome is key for dictating the way a tissue heals
• if left uncontrolled can damage implant & tissues

Question 7

Describe the role of lymphocytes in biocompatibility

Answer 7

• chronic inflammatory cells
• remove foreign molecules persistent throughout the body
• potential role in material destruction & tissue healing

Question 8

Describe the role of fibroblasts in biocompatibility

Answer 8

• ‘matrix secreting machines’ omnipresent in many tissues
• secrete abundant extra cellular matric to secure implant in place

Question 9

Describe the role of mast cells in biocompatibility

Answer 9

• tissue resident cells which act as hubs to direct inflammation at the site of tissue injury through secretion of huge number of cytokines & inflammatory mediators

Question 10

Describe the role of tissue specific cells in biocompatibility

Answer 10

• osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat)
• all needed to perform regenerative roles in devicwe specific applications

Question 11

Define Cell infiltration

Answer 11

the extent to which cells have gotten inside of an impant, and what sort of cells they are

Question 12

Define Encapsulation

Answer 12

• the manner in which the body has ‘sealed’ the implant from the tissue environment around it

Question 13

Define resorption/degeneration

Answer 13

how well the implant retains in structural integrity over time

Question 14

Define Haemorrhage

Answer 14

• the presence of red cells which arent contained within the vasculature

Question 15

Describe Foreign Body Giant cells

Answer 15

• specialised immune cells which engulf macroscopic materials

Question 15

What are often found at implant sites ?

Answer 15

• polymorphonuclear leukocytes

Question 16

Define Vascularisation

Answer 16

provides nutrient influx & waste efflux to/from an implant

Question 16

Describe Oedema

Answer 16

• presence of excessive interstitial fluid withing a tissue space

Question 17

What can long term persistance of Lymphocytes indicate?

Answer 17

• implant failure

Question 18

Describe ISO10993

Answer 18

• set of standard for evaluation the biocompatibilty of medical devices before clinical trials
• typically presented as ‘pathologic index’
• scoring system 0-4 presence =4 abscence =0

Question 19

Describe Napthol ASD Chloroacetate Esterase

Answer 19

• also known as ‘Leders stain’
• esterases found in leukocytes react with defined molecules to produce a coloured product
• naphthol couples with fast red violet LB to create a coloured deposit at site of enzyme activity

Question 19

What is the most common histological method for identifying bone ?

Answer 19

• Von Kossa Staining
• it doesn’t directly interact with calcium

Question 20

Describe Von Kossa Staining

Answer 20

• uses solution of silver nitrate to displace calcium ions from calcium containing compounds in the ECM
• silver compounds are then photochemically degraded into silver atoms by exposure to light
• result is a black precipitate where calcium was present

Question 21

What diseases can Von Kossa indicate?

Answer 21

• demonstrate the broken-down bone trabeculae leading to brittleness we associate with osteoporosis
• can indicate rickets if collagenous material is amongst the black stained mineralised matrix

Question 22

Describe Alizarin red stain

Answer 22

- also used to identify presence of bone - interacts directly with calcium ions forming a chelate which remains on the section surface - less specific than Von Kossa - calcified ECM = deep red

Question 23

Describe the protocol for alizarin red stain

Answer 23

- brings sections to distilled water via xylene & ethanol - stain slides with 2% alizarin red for 2mins - shake off excess dye & blot - dehydrate in acetone, clear in xylene & mount in synthetic mounting medium

Question 24

How is alizarin red quantified using a spectrophotometer ?

Answer 24

- stain is dissolved off the section and its absorbance measured - greater the absorbance, the more bone there was in the section

Question 25

What helps cartilage resist compressive forces?

Answer 25

- the extracellular matrix of cartilage contains abundant proteoglycan structures

Question 26

What does Alcian Blue bind to ?

Answer 26

- proteoglycan structures - turning them a blue/turquoise colour

Question 27

Describe Alcian blue stain

Answer 27

- copper containing dye - +ive charge of coppr allows interaction with -ive charged moieties present in glycosaminoglycans & mucopolysaccharides - glcosylated proteins are abundant in ECM of cartilage

Question 28

What is the protocol of Alcian blue stain?

Answer 28

- deparaffinize slides & hydrate to distilled water - stain in alcian blue solution - wash in running water 2mins - rinse in distilled water - counterstain in nuclear fast red solution for 5 mins - wash in running tap for 1min - dehydrate with ethanol gradient - clear in xylene - mount with DPX

Question 29

What are the results of using Alcian Blue stain?

Answer 29

- strongly acidic/sulphated sugar substances = blue - nuclei = pink/red - cytoplasm = pale pink

Question 30

What does combining alcian blue & PAS allow ?

Answer 30

visualisation a larger range of sugars in a tissue

Question 31

What dye combination allow for the relationship between bone & cartilage ?

Answer 31

alcian blue & alizarin red

Question 32

Why is fat commonly used ?

Answer 32

- reconstructive procedures - similar mechanical properties to lots of soft tissue

Question 33

Describe Oil Red O stain

Answer 33

- stains fat droplets present in cells a deep red colour - mechanism of dye action is largely due to lipophilicity - means it interacts with lipid compartments within cells & remain insoluble in aqeuos environment of normal cytoplasm

Question 34

What is the Oil red O stain procedure?

Answer 34

- cut frozen sections, air dry - fix in formalin, wash with running tap water - rinse with 60% isopropanol - stain with freshly prepared oil red o - rinse with 60% iso... - lightly stain nuclei with alum haematoxylin - rinse with distilled water - mount in glycerine jelly

Question 35

What diease can oil red o help visulise ?

Answer 35

- visualises fat droplets which accumulate in hepatocytes during non-alcoholic fatty liver disease