bone regeneration

Approaches For Bone Regeneration

Presented By:Mr. Mahesh A. Marathe.M. Pharm. Sem I (P’ceutics.)Roll No. 516

A Seminar on

Guide:Dr. K. N Gujar.Mrs. V. M. Gambhire.

SINHGAD COLLEGE OF PHARMACY ,VADGAON , PUNE

Mah

esh

A. M

arat

he. 1

6/11

/201

6

2

Bone : They are made up of living cells .

Minerals, vitamins, proteins are helpful for growth and repair bone.

Bone tissue compose of :Harder outer layer (Cortical/compact bone)

Inner spongy layer (trabecular/cancellous bone).

INTRODUCTION

https://www.iofbonehealth.org/introduction-bone-biology-all-about-our-bones,access on 3/11/2016

Mah

esh

A. M

arat

he. 1

6/11

/201

6

3

Bone cells are composed of :

Osteoblasts and osteocytesosteoclastsOsteiodsInorganic mineral salts

Type of Bones :

Woven boneLamellar bone

https://www.iofbonehealth.org/introduction-bone-biology-all-about-our-bones,access on 3/11/2016

Mah

esh

A. M

arat

he. 1

6/11

/201

6

4

Bone regeneration is process which response to any injury like accidental fracture, development of continuous remodelling of bone, disease condition like osteoporosis and after surgery like arthoplasty, etc

Bone regeneration process involves certain processes like hematoma formation, callus formation, callus ossification and bone remodelling

Madhukar R. W., Dilip R . (2013)Bone Regeneration and Repair: Current and Future Aspects,International Journal of Science and Research (IJSR) ,volume 4 (10),pp.351-355

Mah

esh

A. M

arat

he. 1

6/11

/201

6

5

MECHANISM

6

NATURAL BONE HEALING AND REGENERATION

Mah

esh

A. M

arat

he. 1

6/11

/201

6

Mah

esh

A. M

arat

he. 1

6/11

/201

6

7

There are over 15 million fracture cases globally and an estimated 2.2 million bone graft procedures are performed annually to promote fracture healing or to fill defects .

Sometimes the common solutions, such as Autografts & Allografts are not suitable due to the donor site morbidity, disease transmissions, and, for the implants case, failure may occur after transplantation of tissue.

NEED OF BONE REGENERATION

Carolina G., Frederico R.,Paulo R., (2015),A biomechanical approch for bone regeneration inside scaffolds,procedia engineering,volume 4(2016),pp82-89

Mah

esh

A. M

arat

he. 1

6/11

/201

6

8

Complication in Bone

Regeneration

Fracture shortenin

g

Fracture non

unions

Valgus deformity

Mah

esh

A. M

arat

he. 1

6/11

/201

6

9

Due to these complication or deformities at particular site of facture we need mechanical support for that particular area.

Mechanical support /stability of fracture we need some structural backbone for support like, a) cast b) Rodc) External fixatord) Plates & screw

Mah

esh

A. M

arat

he. 1

6/11

/201

6

10

Mah

esh

A. M

arat

he. 1

6/11

/201

6

11

APPROCHES scaffolds

polymeric

Natural

Bone morphogenic proteins (BMPs)

Mesenchymal cells &Growth factors

12

SCAFFOLDINGNatural- or biologic-based materials are taken from biologic-based tissues.

scaffolding act as Extra Cellular Matrix for cells.

Mah

esh

A. M

arat

he. 1

6/11

/201

6

Mah

esh

A. M

arat

he. 1

6/11

/201

6

13

Biodegradability

Regenerative

Biocompatible

Mah

esh

A. M

arat

he. 1

6/11

/201

6

14

Delivery of cells to desired sites,

scaffold should enhance regenerative capability,

Must provide correct geometry to define and maintain space for tissue regeneration.

FUNCTION OF SCAFFOLDING

Rozalia d.,Elna j.,dennis m.,(2011),bone regeneration:current concepts and future directions,journal of cellular and molecular medicine,volume 15(2011),pp719-738

15

POLYMERIC :Material :chitosan, alginate, etc.Delivery system : hydrogels, fibres, thin films

Natural :Material :collagen, elastin, fibrin, etc.Delivery system : Hydrogels

Ceramic :Material :calcium phosphate cementDelivery system :Porous matrix structures

SCAFFOLDING MATERIALS:

Mah

esh

A. M

arat

he. 1

6/11

/201

6

Mah

esh

A. M

arat

he. 1

6/11

/201

6

16

Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines .

Originally discovered by their ability to induce the formation of bone and cartilage.

. Recombinant human BMPs are used in orthopedic applications such as spinal fusions, nonunions, etc

BONE MORPHOGENETIC PROTEINS


Mah

esh

A. M

arat

he. 1

6/11

/201

6

17

BMPs

Induce differentiations towards osteoblast

Potent osteoinductive factor

Induce mitogenesis

of MSCs

Induce osteoproge

nitors


Mah

esh

A. M

arat

he. 1

6/11

/201

6

18

Mesenchymal stem cells (MSCs) : Multipotent stromal cells that made up of different cell like: osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells).

Stromal cells are connective tissue cells that form the supportive structure to bone .

MESENCHYMAL STEM CELLS

Mah

esh

A. M

arat

he. 1

6/11

/201

6

19

MSCs derived from different sites.

Viktor Tollemar a,b,c, Zach J. Collier a,b, Maryam K. ,et.al, Stem cells,(2013), growth factors and scaffolds in craniofacial regenerative medicine .volume 3, pp59

Mah

esh

A. M

arat

he. 1

6/11

/201

6

20

EXAMPLE..

Viktor Tollemar a,b,c, Zach J. Collier a,b, Maryam K. ,et.al, Stem cells,(2013), growth factors and scaffolds in craniofacial regenerative medicine .volume 3, pp59

Yurong C , Juhong Y, Subhas C. Kundu b, Juming Y (2016), Multifunctional nano-hydroxyapatite and alginate/gelatin based sticky gel composites for potential bone regeneration, Materials Chemistry and Physics, pp 1-7

21

CASE STUDY

Objective :To improve the fixations of the implant and implant-bone integration after joint arthroplasty from locally preventing inflammation and promoting the bone regeneration.

Mah

esh

A. M

arat

he. 1

6/11

/201

6

J.H. Yu, X.B. Chu, Y.R. Cai, P.J. Tong, J.M. Yao, Preparation and characterization of antimicrobial nano-hydroxyapatite composites, Mater. Sci. Eng. C 37 (1) (2014) 54e59.

Mah

esh

A. M

arat

he. 1

6/11

/201

6

22

Synthesis of drug loaded nanohydroxyapatite (nHAP)50ml CaCl2 solution in 150ml of silk sericin solution

Add 50ml Na2HPO4 dropwise in above solution

pH should be made 10 by using 1M NaOH solution

Precipitates collected after 2 hours by centrifugation

Lyophilized to obtain nHAP particles

50oC with agitation30 min

Rinse with ethyl alcohol (3 times)

J.H. Yu, X.B. Chu, Y.R. Cai, P.J. Tong, J.M. Yao, Preparation and characterization of antimicrobial nano-hydroxyapatite composites, Mater. Sci. Eng. C 37 (1) (2014) 54e59.

Mah

esh

A. M

arat

he. 1

6/11

/201

6

23

Preparation of VAN-loaded nHAP :

100mg nHAP in 2 ml SBF (pH – 7.4)containing 5 mg vancomycin chloride (VAN)

At vacuum, at ambient temperature for 24 hours


Mah

esh

A. M

arat

he. 1

6/11

/201

6

24

PREPARATION COMPOSITE GEL :

Nodrug-loaded nHAP/OSA/GT gel

VAN-loaded nHAP/OSA/GT gel

rhBMP-2 loaded nHAP/OSA/GT gel

Both VAN and rhBMP-2loaded nHAP/OSA/GT gel

HOG VHOG BHOG VBHOG


25

Mah

esh

A. M

arat

he. 1

6/11

/201

6

FOR ANALYSIS OF ANTIBACTERIAL ACTIVITY…

Four gel column (1ml each)

Soak in 5ml SBF(pH7.4) at 370

C resp.

After 1,3,5 &7 days supernantant was collected & mixed with S. aureus bacteria cultured media.

After 24 hr. ,culture forming unit (CFU) evaluated for sustained bacterial effect.

Yurong C , Juhong Y, Subhas C. Kundu b, Juming Y (2016), Multifunctional nano-hydroxyapatite and alginate/gelatin based sticky gel composites for potential bone regeneration, Materials Chemistry and Physics, pp 1-7 26

Mah

esh

A. M

arat

he. 1

6/11

/201

6

MG63 cell bone related cell model

50 microgram composite gel injected into 96-well plates

Use blank as a control

Gel were wash by PBS(pH7.4)

Cells on gel were evaluated by MTT test.

ANALYSIS FOR ANTIBACTERIAL ACTIVITY USING CELL MODEL:


27

The chemical composition of obtained particles were analyzed with XRD.

The total amount of VAN loaded and rhBMP-2 loaded on the nHAP particles was calculated using the weight differential method.The morphology of nHAP was investigated using a TEM.

The dispersed degree of nHAP in VBHOG was observed by Field emission scanning electron microscopy.

The stickiness of the composite was measured by the texture analyzer

ANALYTICAL PROCESS FOR GELM

ahes

h A.

Mar

athe

. 16/

11/2

016

28Mah

esh

A. M

arat

he. 1

6/11

/201

6

ANIMAL STUDY :

3 groups for 45 days.

Male rats of weight 250-350 gm.

15 animals in each group.

3 groups : control , HOG and VBHOG.


29

Mah

esh

A. M

arat

he. 1

6/11

/201

6

PROCESS FOR ANIMAL STUDY

Rat anesthesized with 4% chloral hydrate

On middle part of skull bone defect of 0.2 mm size generated using table electric dental engine

Column sample of HOG & VBHOG was inserted.


30

Mah

esh

A. M

arat

he. 1

6/11

/201

6

HISTOLOGICAL STUDY :Each skull

was fixed in 4% PBS (pH7.4) buffer

formalin

Keep skull for over period of

night

decalcification

Embedded with

paraffin

To obtain thin size

slices

Obtain slices of 3

micrometer

Dyed with heamatoxyli

n eosin

Observe under optical

microscopy


31

RESULT AND DISCUSSION

Mah

esh

A. M

arat

he. 1

6/11

/201

6

XRD & TEM


32Mah

esh

A. M

arat

he. 1

6/11

/201

6

Adsorption rate & cumulative release


Mah

esh

A. M

arat

he. 1

6/11

/201

6

Morphology and stickiness

34Mah

esh

A. M

arat

he. 1

6/11

/201

6

Colony count for determination of antibacterial activity.

MG-63 cells (bone related cells) grown to study the cell proliferation using MTT test


Mah

esh

A. M

arat

he. 1

6/11

/201

6BONE REGENERATION

DURING ANIMAL STUDY

Mah

esh

A. M

arat

he. 1

6/11

/201

6

HISTOLOGICAL STUDY


36

Mah

esh

A. M

arat

he. 1

6/11

/201

6

37

CONCLUSION OF CASE STUDY :

A multifunctional composite with rhBMP-2, antibiotic loaded nano-hydroxyapatite and alginate/gelatin sticky gel formulated . It results in reduce the mobilization of the composite on the fractured bone surface structure. Sticky gel composite shows sustained release action.

The composite shows antimicrobial effect and ability to promote the bone cell proliferation. The composite reduce the inflammation, promotes the formation of new bones and blood vessels in vivo.

Mah

esh

A. M

arat

he. 1

6/11

/201

6

38

CONCLUSIONResearch is ongoing to improve : the mechanical properties and biocompatability of scaffolds to promote osteoblast adhesion, growth and differentiation and allow to bone tissue formation.

Some novel nanotechnology approaches such as Magnetic biohybrid porous scaffolds (acting as crooslinking agent for collage for bone regeneration) guided by external magnetic field.

Injectable scaffold is also novel and eaiser application

Yu J.H., , Y.R. CaCHU X.B., TONG P.J., YAO J.M., Preparation and characterization of antimicrobial nano-hydroxyapatite composites, Mater. Sci. Eng. C 37 (1) (2014) 54-59.

39

Yurong C , Juhong Y, Subhas C. Kundu b, Juming Y (2016), Multifunctional nano-hydroxyapatite and alginate/gelatin based sticky gel composites for potential bone regeneration, Materials Chemistry and Physics, pp 1-7..\Downloads\pharmaceutics seminar\seminar topic.pdf

Mah

esh

A. M

arat

he. 1

6/11

/201

6

REFERENCE

Madhukar R. W., Dilip R . (2013)Bone Regeneration and Repair: Current and Future Aspects, International Journal of Science and Research (IJSR), 4 (10),pp.351-355

R15(2011),pp719-738

40

Viktor Tollemar a,b,c, Zach J. Collier a,b, Maryam K.et.al,(2013) Stem cells, growth factors and scaffolds in craniofacial regenerative medicine .volume 3, pp.59..\Downloads\1-s2.0-S2352304215000653-main.pdf

Xu Y.T, Yu X.X, Gu Z.P., X. ,(2012) study of a novel crosslinking reagent (alginate dialdehyde) for biological tissue fixation, Polym.,pp. 1589-1595.

Carolina G., Frederico R.,Paulo R., (2015),A biomechanical approch for bone regeneration inside scaffolds, procedia engineering,volume 4(2016),pp82-89

Mah

esh

A. M

arat

he. 1

6/11

/201

6

Mah

esh

A. M

arat

he. 1

6/11

/201

6

bone regeneration

Health & Medicine