This document discusses bioprinting of osteochondral tissue, which contains both cartilage and bone regions. It outlines printing strategies using alginate hydrogel reinforced with PLA microfibers to mimic cartilage, and a PCL/HA scaffold to mimic bone's varying stiffness gradients. Mesenchymal stem cells would be seeded and differentiated into osteoblasts and chondrocytes. The objectives are to 3D print a scaffold that mimics the native tissue structure and gradients, and evaluate its ability to regenerate tissue in vivo. Challenges include fully mimicking the varying mechanical properties and spatial organization of the native tissue.

1. Bioprinting of Osteochondral Tissue Equivalent
Presented by :
Pradeep kumar yadav
BIOFABRICATION (BM 4190)
PBL presentation
1

2. 2
OUTLINE
 INTRODUCTION
o Osteochondral Tissue
o Problem and Need
 REVIEW OF LITERATURE
 AIM & OBJECTIVE
 MATERIALS
 3D PRINTING STRATEGY
 METHODOLOGY – Technology and biological characterisation
 IN VIVO APPLICATION
 WHAT WE ACHIEVED Vs. NOT
 CONCLUSION

3. Osteochondral tissue
Ostechondral tissue showing different zones of cartilage Variation in chondrocytes, collagen II, GAGs, stiffness
And zonal growth factors.
Vyas C. et. al, Biomedical Composites, 2017 3
• Osteochondral tissue has complex hierarchical and organisational structure

4. • Osteochondral defects contain damage to both the articular cartilage as well as the underlying
subchondral bone.
• With an aging population and obesity , the natural wear of the cartilaginous
tissue often leads to osteoarthritis, a major cause of osteochondral defects.
~ 30% population worldwide suffers.
• Clinical treatment studies going from late 1970s
• Microfacture, Mosaicplasty- allografts/autografs. - limited “effectiveness”
Need of Osteochondral Tissue Engineering
4
Dr.David Ramey illustration

5. Literature Review
1. Schek R. et. al, Orthod Craniofacial Res 8, 2005
PLA scaffold
for cartilage
2. Levingstone T. et. al., Acta Biomaterialia 32, 2016
Hydroxyapatite
scaffold
For bone.
3. Erisken C. et. al., Biomaterials 29, 2008 5

6. Aims & Objective
• To biofabricate osteochondral tissue which can better mimic various
gradients present in cartilage. Also to incorporate bone region with
porosity gradient expected to mimic stiffness variation between
cortical and cancellous bone regions.
• To evaluate the tissue regeneration capability of printed tissue scaffold
in vivo.
6

7. Materials
• For cartilage : Scaffold of sodium alginate reinforced with PLA microfibers. SA concentration : 2-4%
(this concentration is extrudable, structurally stable and biologically acceptable and produce good
fidelity) (He Y. et. al., Scientific reports, 2016)
• PLA as collagen fibers : PLA stiffness = 2.7 – 16 GPa and collagen fibers’s stiffness 5 – 11.5 GPa,
• Mechanical stiffness of 2.5 % SA crosslinked (He Y. et. al., Scientific reports, 2016) = 200 KPa and
cartilages’s bulk stiffness = 0.1 – 20 MPa)
• For bone : PCL/HA : 40 % HA powder, 60 % PCL pellets (Ding C. et al, Biomaterials 34, 2013).
Figure showing above mentioned composition in the region of bone stiffness.
(Ding C. et al, Biomaterials 34, 2013)
7
• Bone marrow derived MSCs + Growth factors TGFβ-1, IGF-1 and BMP-7

8. Chondrogenic
differentiation
medium
Osteogenic
differentiation
medium
Fibrin glue
Hybrid
Ostochondral
scaffold
Porosity
gradient
Bioprinting strategy
8

9. Method & Technology
• Cartilage : First print PLA framework (Scaffold) using multi head FDM extrusion bioprinter using
different orientation. Simultaneously, with each region being printed coat with Collagen. (e.g. print deep
layer and then first coat with collagen X then continue the printing)
• After printing prepare three flasks containing alginate mixture and pour it one by one simultaneously
crosslink with CaCl2. Put the scaffold in chondrogenic differentiation medium and culture the cells for 2-
3 weeks in incubator.
• Simultaneously, use multihead FDM 3d printer to print bone scaffold with variable porosity gradient to
mimic stiffness variation from cortical to spongy bone. Then, coat it with Collagen I and seed with
MSCs, keep it for 2-3 weeks prior to in vivo implantation in incubator with osteogenic differentiation
medium.
• Take out both the scaffold and glue it with fibrin. For in vivo, implant it in rat model. For in vitro this
study maybe carried out for 1 month in incubator and dynamic bioreactor can be used to stimulate
chondrocytes and osteocytes activity.
9

10. Biological characterization
• Cell viability/biocompatibility : Live/Dead Cell Double Staining Calcein-AM /PI and
DAPI staining
• Osteogenic marker : ALP staining and Alizarin red staining
• Chondrogenic marker : Alcian blue
• Gross tissue : H & E, Goldner’s trichrome
• Immunohistochemical analysis : (Col I, osteopontin, osteocalcin for bone), Col II for
cartilage
• microcomputed tomography (µCT) analysis for bone mineralization
10

11. Structure & Function attainable
• Cartilage region : Collagen fiber orientation, zone specific growth factors and chondrocyte density may
enhance differentiation of MSCs to chondrocytes. The fiber reinforced cartilage helps in enhancing
stiffness and hence the scaffold doesn’t suffer from scaffold breakdown in in vivo application.
PCL fiber reinforced SA hydrogel enhanced
stiffness by 15 fold.
Visser J et al, Nature communication 6, 2015
Col II expression significantly enhanced in L1/superficial
Region due to fiber orientation.
Moeinzadeh S. et al, Biomaterials 92, 2016
11

12. • Bone region : PCL/HA is a superior osteoinductive material for in vivo application
PCL/HA demonstrating in vivo bone formation showing
osteopontin, osteocalcin and Collagen I expression post
implantation (10 weeks)
Ding C. et al, Biomaterials 34, 2013
12
Structure & Function attainable
 Graded scaffold the structural interface closely resemble the native environment of the natural tissue
Alizarin red staining for calcium depositions
chondrogenic part and in osteogenic part.
Fedorovich N. et. al, Tissue Engineering Part
C 18, 2012

13. Feasibility for in vivo application
 Comparison among alginate, agarose, PEGMA and GelMA
showing, superior capability of alginate for Cartilage formation
evidenced by GAG and Col II. Daly A. et. al, Biofabrication 8,
2016
 Col II expression, in vivo post
implantation, using SA as scaffold material
 PCL/HA demonstrating in vivo bone formation showing osteopontin,
osteocalcin and collagen I expression post implantation (10 weeks)
Ding C. et al, Biomaterials 34, 2013
13
 Histology (HE staining) of the graft illustrates
heterogeneous tissue formation
Fedorovich N. et. al, Tissue Engineering Part C 18,
2012

14. What was Not attainable ?
• Stiffness of different regions not attainable (Using the same material (SA) for
cartilage, however, variation of concentration of SA may demonstrate stiffness
variation but it can also affect cell viability and its functions)
• Spatial control over chondrocytes not attainable (We are not 3d printing the
alginate)
• Nano fibers of PLA not attainable (minimum diameter of PLA fiber is possible
upto 108 micron (33 gauge needle)) but collagen fiber size range is 20-50 nm (
superficial zone) and 110 nm (deep zone).
• Intact subchondral region and cartilage region not attainable (We are basically
using biological glue or suture)
14

15. Conclusion
• Using 3D biofabrication method, feasibility to construct fiber reinforced hydrogel
scaffolds, with biomimicry of zonal variation of cells, growth factors and collagen
fiber variation in cartilage scaffold was demonstrated.
• The material chosen for both the regions are expected to support cartilage and
bone formation in vitro as well as in vivo.
• The porosity gradient in bone scaffold expected to mimic the stiffness variation of
cortical and spongy bone regions.
15

16. 16
References
1. Vyas, C., Poologasundarampillai, G., Hoyland, J. and Bartolo, P., 2017. 3D printing of biocomposites for osteochondral
tissue engineering. In Biomedical Composites (Second Edition) (pp. 261-302).
2. Ding, C., Qiao, Z., Jiang, W., Li, H., Wei, J., Zhou, G. and Dai, K., 2013. Regeneration of a goat femoral head using a
tissue-specific, biphasic scaffold fabricated with CAD/CAM technology. Biomaterials, 34(28), pp.6706-6716.
3. Schek, R.M., Taboas, J.M., Segvich, S.J., Hollister, S.J. and Krebsbach, P.H., 2004. Engineered osteochondral grafts
using biphasic composite solid free-form fabricated scaffolds. Tissue Engineering, 10(9-10), pp.1376-1385.
4. Levingstone, T.J., Thompson, E., Matsiko, A., Schepens, A., Gleeson, J.P. and O’Brien, F.J., 2016. Multi-layered
collagen-based scaffolds for osteochondral defect repair in rabbits. Acta biomaterialia, 32, pp.149-160.
5. Erisken, C., Kalyon, D.M. and Wang, H., 2008. Functionally graded electrospun polycaprolactone and β-tricalcium
phosphate nanocomposites for tissue engineering applications. Biomaterials, 29(30), pp.4065-4073.
6. Visser, J., Melchels, F.P., Jeon, J.E., Van Bussel, E.M., Kimpton, L.S., Byrne, H.M., Dhert, W.J., Dalton, P.D.,
Hutmacher, D.W. and Malda, J., 2015. Reinforcement of hydrogels using three-dimensionally printed
microfibres. Nature communications, 6, p.6933.

17. 7. Moeinzadeh, S., Shariati, S.R.P. and Jabbari, E., 2016. Comparative effect of physicomechanical and biomolecular
cues on zone-specific chondrogenic differentiation of mesenchymal stem cells. Biomaterials, 92, pp.57-70.
8. Fedorovich, Natalja E et al. “Biofabrication of osteochondral tissue equivalents by printing topologically defined,
cell- laden hydrogel scaffolds” Tissue engineering. Part C, Methods vol. 18,1 (2011): 33-44.
9. Daly, A.C., Critchley, S.E., Rencsok, E.M. and Kelly, D.J., 2016. A comparison of different bioinks for 3D bioprinting
of fibrocartilage and hyaline cartilage. Biofabrication, 8(4), p.045002.
10. Nukavarapu, S.P. and Dorcemus, D.L., 2013. Osteochondral tissue engineering: current strategies and
challenges. Biotechnology advances, 31(5), pp.706-721.
11. Peng, X.B., Zhang, Y., Wang, Y.Q., He, Q. and Yu, Q., IGF‐1 and BMP‐7 synergistically stimulate articular cartilage
repairing in the rabbit knees by improving chondrogenic differentiation of bone‐marrow mesenchymal stem cells. Journal
of Cellular Biochemistry.
References

18. Thank you
Any Questions ?
18