project on bone ingrowth and coverage...protocols in histotechniques: stereology grids it provide an...

PROJECT ON BONE INGROWTH AND

COVERAGEME 6505 - Engineering Materials in Medicine

Presented byGroup III

TEAM MEMBERS

Aravind Baskar

Cao Jiawei

Debirupa Mitra

Karthik Mamandur

Gopalakrishna

Lew Maan Tarng

Naresh Kumar Thanigaivel

Steffie Mano

Tekumalla Venkata Rama

Lakshmi Sravya

Wang Jiahui

Wang Yuzhe

OUTLINE

Introduction

Need for bone implants

Commercial implant materials

Surgical procedure of implants

Project

Measuring techniques and grids

Core project results explanation

Results and discussion

Mechanism of bone integration

Bone remodelling

Bone coverage with time

Others computation methods

PRESENT TO FUTURE

NEED FOR BONE IMPLANTS

Customer Needs

Aging population; expanding pool of

younger patients (minimally invasive

procedures)

Autograft, allograft, bone substitutes

Substitutes introduced due to quantity

limitation and chronic pain of the donor site

More desirable due to less operation time

Company Needs

Increasing customers

Shift to more expensive

demineralized bone matrix

$2.5 billion market, ↑

COMMERCIALLY AVAILABLE IMPLANTSPRODUCT COMPANY COMPOSITION USE PROPERTIES

ProOsteon® 200R

BiometThin coralline-derived HA layer on Calcium carbonate matrix Bone void filler

BioresorbableOsteoconductive

ChronOsTM DePuySynthes

Pure β-TCP

MasterGraft® Putty

MedtronicBiphasic Calcium Phosphate (15% HA, 85% β-TCP) & Type 1 bovine Collagen

Bone void filler/graft extender

Vitoss® BA Stryker70% β-TCP, 20% collagen, 10% bioactive glass

Bone void filler

Actifuse® ABX BaxterSilicon (0.8% by weight) substituted calcium phosphate

BioresorbableOsteoconductive

SURGICAL PROCEDURE OF IMPLANTS

SuturingInsertionHole on animal part

• E.g. Femoral Condye with 4mm diameter and 7mm depth

Sterilization of Bone implant material

• @ 150°C for 4hrs

Animal

• E.g. Rabbit

SURGICAL PROCEDURE OF IMPLANTS

Ref: [1] Patel, N., Best, S. M., Bonfield, W., Gibson, I. R., Hing, K. A., Damien, E., & Revell, P. A. (2002). A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules. Journal of Materials Science: Materials in Medicine, 13(12), 1199-1206.

HISTOLOGYHistology is an essential tool of biology and medicine. The study of tissues and organs through the

examination of the microscopical architecture of tissues and the relationship between the

different types of cells and tissue types found within tissues and organs.

Receipt and Identification

Labelling of Specimen

Fixation

Dehydration

Clearing

Impregnation

Embedding

Section Cutting

Staining

Mounting

Protocols in Histotechniques:

Stereology Grids

It Provide an established set of tools for the stereological estimation of bone perimeter, bone area, and bone surface.

Available Grids

Merz Grids: Bone Perimeter and Area Estimation

Point Grid: Quick Bone Area Estimation

XY Lines Grid: Systematic Random Sampling

Weibel Grids: Bone Perimeter and Area Estimation

Cycloid Grids: Bone Surface Estimation

How and Why do they do it?

The grids are super-imposed onthe images of the slides todetermine the quantitativeparameters of tissue structureon sections.

It includes the estimation ofvolumetric ratios, surface areas,surface-to-volume ratios,thicknesses of tissue or cellsheets and the number ofstructures.

HISTOLOGY

MEASUREMENT TECHNIQUES AND GRIDS

Ref. : Weibel & Elias, 1967; Weibel, E. R., Kistler, G. S., & Scherle, W. F. (1966).

MEASUREMENT TECHNIQUES AND GRIDS

Method of estimating

surface area:

Method of estimating volumetric

composition of tissues:

Method of estimating surface to

volume ratio:

Ref. : Weibel & Elias, 1967; Weibel, E. R., Kistler, G. S., & Scherle, W. F. (1966).

SOLUTION METHODOLOGY

The given micrographs were studied according to the

techniques mentioned in the project description and the

results were analysed and compared with the literature / ref.

materials. From the results and the literature review the final

solutions were arrived.

COMPUTATION METHOD

MICROGRAPH - I

Measurement of Bone Ingrowth

Zone HBI AZBI Avg.

I 6 14%

21%II 8 19%

III 14 33%

IV 8 19%

MICROGRAPH - II

Measurement of Bone Ingrowth

Zone HBI AZBI Avg.

I 8 19%

32%II 9 21%

III 16 38%

IV 20 48%

MICROGRAPH - III

Measurement of Bone Coverage

Zone HBC HIS ZBC Avg.

I 10 29 26%

52%II 23 14 62%

III 19 11 63%

IV 22 13 63%

MICROGRAPH - IV

Measurement of Bone Coverage

Zone HBC HIS ZBC Avg.

I 24 3 89%

83%II 16 1 94%

III 10 6 63%

IV 26 6 81%

RESULTS & DISCUSSION

Parameter Micrographs – I & III Micrograph – II & IV Therefore, over time, a

Si-HA implant will have

significantly enhanced

bonding with the human

system than a HA

implant.

Bone Ingrowth 21% 32%

Bone Coverage 52% 83%

From the data, it is inferred that micrographs I & III

belong to HA and micrographs II & IV belong to Si-HA

as a result of better bone ingrowth and coverage.

REASONS FOR INCREASED BONE INGROWTH IN SI-HA IMPLANTS

FORCE VIEW

Incorporation of Si into the HA lattice results in an increased Van der Waal’s

interaction and an increase in the negative surface charge density in comparison

to the unsubstituted HA.

Average adhesion force versus surface charge for HA and Si-HA surfaces

Ref: J. Vandiver, D. Dean, N. Patel, C. Botelho, S. Best, J. D. Santos, M. A. Lopes, W. Bonfield, and C. Ortiz, “Silicon addition to hydroxyapatite increases nanoscale electrostatic, van der Waals, and adhesive interactions.,” J. Biomed. Mater. Res. A, vol. 78, no. 2, pp. 352–63, Aug. 2006.

REASONS FOR INCREASED BONE INGROWTH IN SI-HA IMPLANTS

Day 1 Day 3 Day 42

Coating morphology on Day 1, Day 3 and Day 42.

Ref: E. S. Thian, J. Huang, S. M. Best, Z. H. Barber, R. A. Brooks, N. Rushton, and W. Bonfield, “The response of osteoblasts to nanocrystalline silicon-substituted hydroxyapatite thin films.,” Biomaterials, vol. 27, no. 13, pp. 2692–8, May 2006.

MATERIALS VIEW

Silicon causes the tetrahedral distortion and disorder at the hydroxyl site

which decreases stability of the apatite structure and increases the bio reactivity

by forming a carbonate containing apatite layer which favours cell adhesion.

REASONS FOR INCREASED BONE INGROWTH IN SI-HA IMPLANTS

Water spreading behavior on the surface of Si-HA coatings

Contact angle measurements of HA and Si-HA surfaces

Ref: J. Vandiver, D. Dean, N. Patel, C. Botelho, S. Best, J. D. Santos, M. A. Lopes, W. Bonfield, and C. Ortiz, “Silicon addition to hydroxyapatite increases nanoscale electrostatic, van der Waals, and adhesive interactions.,” J. Biomed. Mater. Res. A, vol. 78, no. 2, pp. 352–63, Aug. 2006.M. A. Surmeneva, A. Kovtun, A. Peetsch, S. N. Goroja, A. A. Sharonova, V. F. Pichugin, I. Y. Grubova, A. A. Ivanova, A. D. Teresov, N. N. Koval, V. Buck, A. Wittmar, M. Ulbricht, O. Prymak, M. Epple, and R. A. Surmenev, “Preparation of a silicate-containing hydroxyapatite-based coating by magnetron sputtering: structure and osteoblast-like MG63 cells in vitro study,” RSC Adv., vol. 3, no. 28, p. 11240, Jun. 2013.

SURFACE VIEW

Si-HA has a lower contact angle and is more hydrophilic than HA.

BONE COVERAGE CHANGE WITH TIME

Ref: Patel N, Brooks RA, Clarke MT, Lee PMT, Rushton N, Gibson IR, et al. In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model. J Mater Sci. 2005;16: 429-40

MECHANISM OF BONE INTEGRATION Implanted granules dissolve to release Ca2+ and

PO43- ions into the ECM

Cell signaling is triggered due to formation ofapatite-like surface layer

Proteins will deposit onto implant surface.

Following initial protein adsorption, cellrecruitment is stimulated over the implantsurface

Cells adhere over implant surface

Cells proliferate and differentiate, accompaniedby the mineralization of calcium phosphate

Interlock bonding between existing bone andimplant material

Consequently implanted region is filled upsuccessfully leading to bone integration

BONE REMODELLING

Ref: A presentation on “ Bone Histology and Histopathology for Clinicians “by Stephen F. Hodgson.

ADDITIONAL COMPUTATIONAL METHOD

Image processing using MATLAB

For measurement of bone ingrowth

Turn original image to gray image

Find the two threshold values by

‘multi-thresh’ function

Label the 3 groups determined by the threshold values

Find the number of pixels which belong

to the new bone

Calculate the

percentage of bone

ingrowth

ADDITIONAL COMPUTATIONAL METHOD