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
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
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
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
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
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
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
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
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
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
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
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:
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
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.
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
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.
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
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
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
31
RESULT AND DISCUSSION
Mah
esh
A. M
arat
he. 1
6/11
/201
6
XRD & TEM
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
32Mah
esh
A. M
arat
he. 1
6/11
/201
6
Adsorption rate & cumulative release
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 33
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
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 35
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
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
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