Analysis of the in vivo regeneration capability of any tissue-engineered biomaterial is necessary once it shows potential characteristics during in vitro studies. Thus, we applied polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogel on critical-sized cranial bone defects in wistar rats for examining the comparative bone regeneration of cryogel-treated and nontreated defects over a period of 4 weeks. An in-depth analysis was performed from macroscopic level till the gene level. Bone regeneration in cryogel-treated defects was clearly evident from the results, whereas the nontreated group did not show any defect healing except at few peripheral areas. At the macroscopic level, micro-computed tomography analysis revealed new bone formation. This was further confirmed at the cellular level, wherein, new bone formation was demonstrated by hematoxylin and eosin staining. Osteoblastic differentiation was further validated by immunohistological staining of runt-related transcription factor-2 (Runx-2) protein and via calcium-phosphate crystal formation after 2 weeks through scanning electron microscopy and energy dispersive X-ray spectroscopy. Finally, at the gene level, real-time PCR analysis confirmed the mRNA expression of osteoblastic markers, that is, runx-2, collagen type I (Col I), alkaline phosphatase (ALP), and osteocalcin (OCN). Therefore, the results of in vivo cranial defect model studies suggest that PTAC biocomposite cryogels can show suitable potential for human bone regeneration.
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References
1.
SchmitzJ.P., and HollingerJ.O.The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res, 205, 299, 1986.
LangerR., and VacantiJ.P.Tissue engineering. Science, 260, 920, 1993.
4.
PutnamJ.A., and MooneyJ.D.Tissue engineering using synthetic extracellular matrices. Nat Med, 7, 824, 1996.
5.
LangerR.Tissue engineering: a new field and its challenges. Pharm Res, 14, 840, 1997.
6.
SoucacosP.N., JohnsonE.O., and BabisG.An update on recent advances in bone regeneration. Injury, 39Suppl 2, S1, 2008.
7.
SalgadoA.J., CoutinhoO.P., and ReisR.L.Bone tissue engineering: state of the art and future trends. Macromol Biosci, 4, 743, 2004.
8.
KneserU., SchaeferD.J., PolykandriotisE., and HorchR.E.Tissue engineering of bone: the reconstructive surgeon's point of view. J Cell Mol Med, 10, 7, 2006.
9.
DrosseI., VolkmerE., CapannaR., BiaseP.D., MutschlerW., and SchiekerM.Tissue engineering for bone defect healing: an update on a multi-component approach. Injury, 39, S9, 2008.
10.
WoodruffM.A., LangeC., ReichertJ., BernerA., ChenF., FratzlP., SchantzJ.T., and HutmacherD.W.Bone tissue engineering: from bench to bedside. Mater Today, 15, 430, 2012.
11.
MishraR., and KumarA.Inorganic-organic biocomposite cryogels for regeneration of bony tissues. J Biomater Sci Polym Ed, 22, 2107, 2011.
12.
UedaK., ObaS., OmiyaY., and OkadaM.Cranial-bone defects with depression deformity treated with ceramic implants and free-flap transfers. Br J Plast Surg, 54, 403, 2001.
13.
LundgrenD., NymanS., MathisenT., IsakssonS., and KlingeB.Guided bone regeneration of cranial defects, using biodegradable barriers: an experimental pilot study in the rabbit. J Craniomaxillofac Surg, 20, 257, 1992.
14.
CuiL., LiuB., LiuG., ZhangW., CenL., SunJ., YinS., LiuW., and CaoY.Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model. Biomaterials, 28, 5477, 2007.
15.
BurdickJ.A., FrankelD., DernellW.S., and AnsethK.S.An initial investigation of photocurable three-dimensional lactic acid based scaffolds in a critical-sized cranial defect. Biomaterials, 24, 1613, 2003.
16.
RuhéP.Q., Kroese-DeutmanH.C., WolkeJ.G.C., SpauwenP.H.M., and JansenJ.A.Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials, 25, 2123, 2004.
17.
ChenT.M., YaoC.H., WangH.J., ChouG.H., LeeT.W., and LinF.H.Evaluation of a novel malleable, biodegradable osteoconductive composite in a rabbit cranial defect model. Mater Chem Phys, 55, 44, 1998.
18.
ChesmelK.D., BrangerJ., WertheimH., and ScarboroughN.Healing response to various forms of human demineralized bone matrix in athymic rat cranial defects. J Oral Maxillofac Surg, 56, 857, 1998.
19.
LeiggenerC.S., CurtisR., MüllerA.A., PflugerD., GogolewskiS., and RahnB.A.Influence of copolymer composition of polylactide implants on cranial bone regeneration. Biomaterials, 27, 202, 2006.
20.
SroujiS., FromiguéO., VaudinP., Ben-DavidD., LivneE., KuhnG., MullerR., and MarieP.J.Repair of critical-size mouse cranial and long bone defects using human adult mesenchymal stromal cells expressing integrin alpha 5. Bone, 48, S170, 2011.
21.
ChangS.C., ChuangH., ChenY.R., YangL.C., ChenJ.K., MardiniS., ChungH.Y., LuY.L., MaW.C., and LouJ.Cranial repair using BMP-2 gene engineered bone marrow stromal cells. J Surg Res, 119, 85, 2004.
22.
DupoirieuxL., PohlJ., HankeD., and PourquierA.preliminary report on the effect of dimeric rhGDF-5 and its monomeric form rhGDF-5C465A on bone healing of rat cranial defects. J Craniomaxillofac Surg, 37, 30, 2009.
23.
XuS., WuB., and GuoX.BMP2 related oligopeptide as a potential therapeutic agent to accelerate bone formation and bone defect repairing in osteoporotic fracture: Effect on rat cranial bone defects repair. Bone, 47, S368, 2010.
24.
ProloD.J., PedrottiP.W., BurresK.P., and OklundS.Superior osteogenesis in transplanted allogeneic canine skull following chemical sterilization. Clin Orthop Relat Res, 168, 230, 1982.
25.
AnY.H., and FriedmanR.J.Animal models of bone defect repair. In: AnY.H., and FriedmanR.J., eds. Animal Models in Othopaedic Research. Boca Raton: CRC Press, 1998, pp. 241–260.
26.
SzpalskiC., BarrJ., WetterauM., SaadehP.B., and WarrenS.M.Cranial bone defects: current and future strategies. Neurosurg Focus, 29, 1, 2010.
27.
RebueltoM., AmbrosL., WaxmanS., and MontoyaL.Chronobiological study of the pharmacological response of rats to combination ketamine–midazolam. Chronobiol Int, 21, 591, 2004.
28.
NgueguimaF.T., KhanM.P., DonfackJ.H., SiddiquiJ.A., TewariD., NagarG.K., TiwariS.C., TheophileD., MauryaR., and ChattopadhyayN.Evaluation of Cameroonian plants towards experimental bone regeneration. J Ethnopharmacol, 141, 331, 2012.
29.
SharanK., MishraJ.S., SwarnkarG., SiddiquiJ.A., KhanK., KumariR., RawatP., MauryaR., SanyalS. and ChattopadhyayN.A novel quercetin analogue from a medicinal plant promotes peak bone mass achievement and bone healing after injury and exerts an anabolic effect on osteoporotic bone: the role of aryl hydrocarbon receptor as a mediator of osteogenic action. J Bone Miner Res, 26, 2096, 2011.
30.
PfafflM.W., GerstmayerbB., BosiobA., and WindischW.Effect of zinc deficiency on the mRNA expression pattern in liver and jejunum of adult rats: monitoring gene expression using cDNA microarrays combined with real-time RT-PCR. J Nutr Biochem, 14, 691, 2003.
31.
HenchL.L., and PolakJ.M.Third-generation biomedical materials. Science, 295, 1014, 2002.
32.
MishraR., BasuB., and KumarA.Physical and cytocompatibility properties of bioactive glass-polyvinyl alcohol-sodium alginate biocomposite foams prepared via sol-gel processing for trabecular bone regeneration. J Mater Sci Mater Med, 20, 2493, 2009.
33.
KumarA., MishraR., ReinwaldY., and BhatS.CRYOGELS: freezing unveiled by thawing. Mater Today, 13, 42, 2010.
34.
KumarA., and SrivastavaA.Cell separation using cryogel based affinity chromatography. Nat Protoc, 5, 1737, 2010.
35.
JonesJ.R., GentlemanE., and PolakJ.Bioactive glass scaffolds for bone regeneration. Elements, 3, 393, 2007.
36.
MontjoventM.O., MathieuL., SchmoekelH., MarkS., BourbanP.E., ZambelliP.Y., Laurent-ApplegateL.A., and PiolettiD.P.Repair of critical size defects in the rat cranium using ceramic-reinforced PLA scaffolds obtained by supercritical gas foaming. J Biomed Mater Res A, 83, 41, 2007.
37.
ChesnuttB.M., YuanY., BuddingtonK., HaggardW.O., and BumgardnerJ.D.Composite chitosan/nano-hydroxyapatite scaffolds induce osteocalcin production by osteoblasts in vitro and support bone formation in vivo. Tissue Eng Part A, 15, 2571, 2009.
