Postnatal ex vivo rat model for longitudinal bone growth investigations
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摘要
Background: Chondrocytes in the growth plate(GP) undergo increases in volume during different cascades of cell differentiation during longitudinal bone growth. The volume increase is reported to be the most significant variable in understanding the mechanism of long bone growth.Methods: Forty-five postnatal Sprague-Dawley rat pups, 7-15 days old were divided into nine age groups(P7-P15). Five pups were allocated to each group. The rats were sacrificed and tibia and metatarsal bones were harvested. Bone lengths were measured after 0, 24, 48, and 72 hours of ex vivo incubation. Histology of bones was carried out, and GP lengths and chondrocyte densities were determined.Results: There were significant differences in bone length among the age groups after 0 and 72 hours of incubation. Histological sectioning was possible in metatarsal bone from all age groups, and in tibia from 7-to 13-day-old rats. No significant differences in tibia and metatarsal GP lengths were seen among different age groups at 0 and 72 hours of incubation. Significant differences in chondrocyte densities along the epiphyseal GP of the bones between 0 and 72 hours of incubation were observed in most of the age groups.Conclusion: Ex vivo growth of tibia and metatarsal bones of rats aged 7-15 days old is possible, with percentage growth rates of 23.87 ± 0.80% and 40.38 ± 0.95%measured in tibia and metatarsal bone, respectively. Histological sectioning of bones was carried out without the need for decalcification in P7-P13 tibia and P7-P15 metatarsal bone. Increases in chondrocyte density along the GP influence overall bone elongation.
Background: Chondrocytes in the growth plate(GP) undergo increases in volume during different cascades of cell differentiation during longitudinal bone growth. The volume increase is reported to be the most significant variable in understanding the mechanism of long bone growth.Methods: Forty-five postnatal Sprague-Dawley rat pups, 7-15 days old were divided into nine age groups(P7-P15). Five pups were allocated to each group. The rats were sacrificed and tibia and metatarsal bones were harvested. Bone lengths were measured after 0, 24, 48, and 72 hours of ex vivo incubation. Histology of bones was carried out, and GP lengths and chondrocyte densities were determined.Results: There were significant differences in bone length among the age groups after 0 and 72 hours of incubation. Histological sectioning was possible in metatarsal bone from all age groups, and in tibia from 7-to 13-day-old rats. No significant differences in tibia and metatarsal GP lengths were seen among different age groups at 0 and 72 hours of incubation. Significant differences in chondrocyte densities along the epiphyseal GP of the bones between 0 and 72 hours of incubation were observed in most of the age groups.Conclusion: Ex vivo growth of tibia and metatarsal bones of rats aged 7-15 days old is possible, with percentage growth rates of 23.87 ± 0.80% and 40.38 ± 0.95%measured in tibia and metatarsal bone, respectively. Histological sectioning of bones was carried out without the need for decalcification in P7-P13 tibia and P7-P15 metatarsal bone. Increases in chondrocyte density along the GP influence overall bone elongation.
Background: Chondrocytes in the growth plate(GP) undergo increases in volume during different cascades of cell differentiation during longitudinal bone growth. The volume increase is reported to be the most significant variable in understanding the mechanism of long bone growth.Methods: Forty-five postnatal Sprague-Dawley rat pups, 7-15 days old were divided into nine age groups(P7-P15). Five pups were allocated to each group. The rats were sacrificed and tibia and metatarsal bones were harvested. Bone lengths were measured after 0, 24, 48, and 72 hours of ex vivo incubation. Histology of bones was carried out, and GP lengths and chondrocyte densities were determined.Results: There were significant differences in bone length among the age groups after 0 and 72 hours of incubation. Histological sectioning was possible in metatarsal bone from all age groups, and in tibia from 7-to 13-day-old rats. No significant differences in tibia and metatarsal GP lengths were seen among different age groups at 0 and 72 hours of incubation. Significant differences in chondrocyte densities along the epiphyseal GP of the bones between 0 and 72 hours of incubation were observed in most of the age groups.Conclusion: Ex vivo growth of tibia and metatarsal bones of rats aged 7-15 days old is possible, with percentage growth rates of 23.87 ± 0.80% and 40.38 ± 0.95%measured in tibia and metatarsal bone, respectively. Histological sectioning of bones was carried out without the need for decalcification in P7-P13 tibia and P7-P15 metatarsal bone. Increases in chondrocyte density along the GP influence overall bone elongation.
引文
1.Mackie EJ,Tatarczuch L,Mirams M.The skeleton:a multi-functional complex organ:the growth plate chondrocyte and endochondral ossification.J Endocrinol.2011;211:109-121.
2.Tsang KY,Chan D,Cheah KS.Fate of growth plate hypertrophic chondrocytes:death or lineage extension?Dev Growth Differ.2015;57:179-192.
3.Dowthwaite GP,Bishop JC,Redman SN,et al.The surface of articular cartilage contains a progenitor cell population.J Cell Sci.2004;117:889-897.
4.Loqman MY,Bush PG,Farquharson C,Hall AC.Suppression of mammalian bone growth by membrane transport inhibitors.J Cell Biochem.2013;114:658-668.
5.Staines KA,Pollard AS,McGonnell IM,Farquharson C,Pitsillides AA.Cartilage to bone transitions in health and disease.J Endocrinol.2013;219:R1-R12.
6.Sun MM,Beier F.Chondrocyte hypertrophy in skeletal development,growth,and disease.Birth Defects Res C Embryo Today.2014;102:74-82.
7.Studer D,Millan C,?ztürk E,Maniura-Weber K,Zenobi-Wong M.Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells.Eur Cells Mater.2012;24:118-135.
8.Chagin AS,Karimian E,Sundstr?m K,Eriksson E,S?vendahl L.Catch-up growth after dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones.J Endocrinol.2010;204:21-29.
9.Sun H,Zang W,Zhou B,Xu L,Wu S.DHEA suppresses longitudinal bone growth by acting directly at growth plate through estrogen receptors.Endocrinology.2011;152:1423-1433.
10.Abubakar AA,Noordin MM,Azmi TI,Kaka U,Loqman MY.The use of rats and mice as animal model in ex vivo bone growth and development studies.Bone Joint Res.2016;5:610-618.
11.Loqman MY,Bush PG,Farquharson C,Hall AC.A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions:importance of fixative osmolarity for maintaining morphology.Eur Cell Mater.2010;19:214-227.
12.Marino S,Staines KA,Brown G,Howard-Jones RA,Adamczyk M.Models of ex vivo explant cultures:applications in bone research.BoneKey Rep.2016;5:818.
13.Houston DA,Staines KA,MacRae VE,Farquharson C.Culture of murine embryonic metatarsals:a physiological model of endochondral Ossification.J Vis Exp.2016;118:e54978.
14.Rahman H,Qasim M,Schultze FC,Oellerich M,Asif AR.Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome.Proteome Science.2011;9:71.
15.M?rtensson K,Chrysis D,S?vendahl L.Interleukin-1βand TNF-αact in synergy to inhibit longitudinal growth in fetal rat metatarsal bones.J Bone Mine Res.2004;19:1805-1812.
16.Dettmeyer RB.Staining Techniques and Microscopy.In:Dettmeyer RB,ed.Forensic histopathology fundamental and perspectives.1st edn.Berlin,Heidelberg:Springer-Verlang;2011:17-35.
17.Pastoureau PC,Hunziker EB,Pelletier JP.Cartilage,bone and synovial histomorphometry in animal models of osteoarthritis.Osteoarthritis Cartilage.2010;18:S106-S112.
18.Schmitz N,Laverty S,Kraus VB,Aigner T.Basic methods in histopathology of joint tissues.Osteoarthritis Cartilage.2010;18:S113-S116.
19.Alvarez J,Balbín M,Santos F,Fernández M,Ferrando S,López JM.Different bone growth rates are associated with changes in the expression pattern of types II and X collagens and collagenase 3 in proximal growth plates of the rat tibia.J Bone Mine Res.2000;15:82-94.
20.Wilsman NJ,Bernardini ES,Leiferman E,Noonan K,Farnum CE.Age and pattern of the onset of differential growth among growth plates in rats.J Orthop Res.2008;26:1457-1465.
21.Bush PG,Pritchard M,Loqman MY,Damron TA,Hall AC.A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate.J Bone Mine Res.2010;25:1594-1603.
22.Shapiro IM,Adams CS,Freeman T,Srinivas V.Fate of the hypertrophic chondrocyte:microenvironmental perspectives on apoptosisand survival in the epiphyseal growth plate.Birth Defects Res CEmbryo Today.2005;75:330-339.
23.Amini S,Veilleux D,Villemure I.Three-dimensional in situ zonal morphology of viable growth plate chondrocytes:a confocal microscopy study.J Orthop Res.2010;29:710-717.
24.Tivesten A,Movérare-Skrtic S,Chagin A,et al.Additive protective effects of estrogen and androgen treatment on trabecular bone in ovariectomized rats.J Bone Mine Res.2004;19:1833-1839.
25.Weise M,De-Levi S,Barnes KM,Gafni RI,Abad V,Baron J.Effects of estrogen on growth plate senescence and epiphyseal fusion.Pro Natl Acad Sci U S A.2001;98:6871-6876.
26.Gruber HE,Ingram JA.Basic Staining Techniques for Cartilage Sections.In:An YH,Martin KL,eds.Handbook of histology methods for bone and cartilage.Totowa,NJ:Humana Press;2003:287-293.
27.Dobie R,Ahmed SF,Staines KA,et al.Increased linear bone growth by GH in the absence of SOCS2 is independent of IGF-1.J Cell Physiol.2015;230:2796-2806.
28.Ballock RT,O'Keefe RJ.Physiology and pathophysiology of the growth plate.Birth Defects Res C Embryo Today.2003;69:123-143.
29.Chung UI.Essential role of hypertrophic chondrocytes in endochonral ossification in bone development.Endocr J.2004;51:19-24.
30.Mackie EJ,Ahmed YA,Tatarczuch L,Chen KS,Mirams M.Endochondral ossification:how cartilage is converted into bone in the developing skeleton.Int J Biochem Cell Biol.2008;40:46-62.
31.Rolian C.Developmental basis of limb length in rodents:evidence for multiple divisions of labor in mechanisms of endochondral bone growth.Evol Dev.2008;10:15-28.
32.Walzer SM,Cetin E,Grubl-Barabas R,et al.Vascularization of primary and secondary ossification centers in the human growth plate.BMC Dev Biol.2014;14:36.
33.Enishi T,Yukata K,Takahashi M,Sato R,Sairyo K,Yasui N.Hypertrophic chondrocytes in the rabbit growth plate can proliferate and differentiate into osteogenic cells when capillary invasion is interposed by a membrane filter.PLoS ONE.2014;9:e104638.
34.Kronenberg HM.Developmental regulation of the growth plate.Nature.2003;423:332-336.
35.Yang L,Tsang KY,Tang HC,Chan D,Cheah KS.Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation.Proc Natl Acad Sci U S A.2014;111:12097-12102.
36.de Crombrugghe B,Lefebvre V,Nakashima K.Regulatory mechanisms in the pathways of cartilage and bone formation.Cur Opin Cell Biol.2001;13:721-727.
37.Farnum CE,Lee R,O'Hara K,Urban JP.Volume increase in growth plate chondrocytes during hypertrophy:the contribution of organic osmolytes.Bone.2002;30:574-581.
38.von Pfeil DJ,DeCamp CE.The epiphyseal plate:physiology,anatomy and trauma.Compend Contin Educ Vet.2009;31:E1-E11.
39.Nilsson O,Baron J.Fundamental limits on longitudinalbone growth:growth plate senescenceand epiphyseal fusion.Trends Endocrinol Metab.2004;15:371-374.
40.Cooper KL,Oh S,Sung Y,Dasari RR,Kirschner MW,Tabin CJ.Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions.Nature.2013;495:375-378.
41.Mohammad KS,Chirgwin JM,Guise TA.Assessing new bone formation in neonatal calvarial organ culture.Methods Mol Biol.2008;455:37-50.
42.Martin EA,Ritman EL,Turner RT.Time course of epiphyseal growth plate fusion in rat tibiae.Bone.2003;32:261-267.
43.Nilsson O,Marino R,De Luca F,Phillip M,Baron J.Endocrine regulation of the growth plate.Horm Res.2005;64:157-165.
44.Shim KS.Pubertal growth and epiphyseal fusion.Ann Pediatr Endocrinol Metab.2015;20:8-12.
45.Wilsman NJ,Farnum CE,Green EM,Lieferman EM,Clayton MK.Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates.J Orthop Res.1996;14:562-572.
46.Gkiatas L,Lykissas M,Kostas-Agnantis L,Korompilias A,Batistatou A,Beris A.Factors affecting bone growth.Am J Orthop(Belle Mead NJ).2015;44:61-67.
47.Locatelli V,Bianchi VE.Effect of GH/IGF-1 on bone metabolism and osteoporsosis.Int J Endocrinol.2014;2014:235060.
48.Giustina A,Mazziotti G,Canalis E.Growth hormone,insulin-Like growth factors,and the skeleton.Endocr Rev.2008;29:535-559.
49.Williams JL,Do PD,Eick JD,Schmidt TL.Tensile properties of the physis vary with anatomic location,thickness,strain rate and age.JOrthop Res.2001;19:1043-1048.
50.Mangiavini L,Merceron C,Schipani E.Analysis of mouse growth plate development.Curr Protoc Mouse Biol.2017;6:67-130.
51.Salic A,Mitchison TJ.A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc Natl Acad Sci U S A.2008;105:2415-2420.
52.Srinivas V,Shapiro IM.The Epiphyseal Growth Plate:the Engine That Drives Bone Elongation.In:Preedy VR,ed.Handbook of growth and growth monitoring in health and disease.New York,NY:Springer;2012:1331-1349.
53.Villemure I,Stokes IA.Growth plate mechanics and mechanobiology.A survey of present understanding.J Biomech.2009;42:1793-1803.
2.Tsang KY,Chan D,Cheah KS.Fate of growth plate hypertrophic chondrocytes:death or lineage extension?Dev Growth Differ.2015;57:179-192.
3.Dowthwaite GP,Bishop JC,Redman SN,et al.The surface of articular cartilage contains a progenitor cell population.J Cell Sci.2004;117:889-897.
4.Loqman MY,Bush PG,Farquharson C,Hall AC.Suppression of mammalian bone growth by membrane transport inhibitors.J Cell Biochem.2013;114:658-668.
5.Staines KA,Pollard AS,McGonnell IM,Farquharson C,Pitsillides AA.Cartilage to bone transitions in health and disease.J Endocrinol.2013;219:R1-R12.
6.Sun MM,Beier F.Chondrocyte hypertrophy in skeletal development,growth,and disease.Birth Defects Res C Embryo Today.2014;102:74-82.
7.Studer D,Millan C,?ztürk E,Maniura-Weber K,Zenobi-Wong M.Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells.Eur Cells Mater.2012;24:118-135.
8.Chagin AS,Karimian E,Sundstr?m K,Eriksson E,S?vendahl L.Catch-up growth after dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones.J Endocrinol.2010;204:21-29.
9.Sun H,Zang W,Zhou B,Xu L,Wu S.DHEA suppresses longitudinal bone growth by acting directly at growth plate through estrogen receptors.Endocrinology.2011;152:1423-1433.
10.Abubakar AA,Noordin MM,Azmi TI,Kaka U,Loqman MY.The use of rats and mice as animal model in ex vivo bone growth and development studies.Bone Joint Res.2016;5:610-618.
11.Loqman MY,Bush PG,Farquharson C,Hall AC.A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions:importance of fixative osmolarity for maintaining morphology.Eur Cell Mater.2010;19:214-227.
12.Marino S,Staines KA,Brown G,Howard-Jones RA,Adamczyk M.Models of ex vivo explant cultures:applications in bone research.BoneKey Rep.2016;5:818.
13.Houston DA,Staines KA,MacRae VE,Farquharson C.Culture of murine embryonic metatarsals:a physiological model of endochondral Ossification.J Vis Exp.2016;118:e54978.
14.Rahman H,Qasim M,Schultze FC,Oellerich M,Asif AR.Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome.Proteome Science.2011;9:71.
15.M?rtensson K,Chrysis D,S?vendahl L.Interleukin-1βand TNF-αact in synergy to inhibit longitudinal growth in fetal rat metatarsal bones.J Bone Mine Res.2004;19:1805-1812.
16.Dettmeyer RB.Staining Techniques and Microscopy.In:Dettmeyer RB,ed.Forensic histopathology fundamental and perspectives.1st edn.Berlin,Heidelberg:Springer-Verlang;2011:17-35.
17.Pastoureau PC,Hunziker EB,Pelletier JP.Cartilage,bone and synovial histomorphometry in animal models of osteoarthritis.Osteoarthritis Cartilage.2010;18:S106-S112.
18.Schmitz N,Laverty S,Kraus VB,Aigner T.Basic methods in histopathology of joint tissues.Osteoarthritis Cartilage.2010;18:S113-S116.
19.Alvarez J,Balbín M,Santos F,Fernández M,Ferrando S,López JM.Different bone growth rates are associated with changes in the expression pattern of types II and X collagens and collagenase 3 in proximal growth plates of the rat tibia.J Bone Mine Res.2000;15:82-94.
20.Wilsman NJ,Bernardini ES,Leiferman E,Noonan K,Farnum CE.Age and pattern of the onset of differential growth among growth plates in rats.J Orthop Res.2008;26:1457-1465.
21.Bush PG,Pritchard M,Loqman MY,Damron TA,Hall AC.A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate.J Bone Mine Res.2010;25:1594-1603.
22.Shapiro IM,Adams CS,Freeman T,Srinivas V.Fate of the hypertrophic chondrocyte:microenvironmental perspectives on apoptosisand survival in the epiphyseal growth plate.Birth Defects Res CEmbryo Today.2005;75:330-339.
23.Amini S,Veilleux D,Villemure I.Three-dimensional in situ zonal morphology of viable growth plate chondrocytes:a confocal microscopy study.J Orthop Res.2010;29:710-717.
24.Tivesten A,Movérare-Skrtic S,Chagin A,et al.Additive protective effects of estrogen and androgen treatment on trabecular bone in ovariectomized rats.J Bone Mine Res.2004;19:1833-1839.
25.Weise M,De-Levi S,Barnes KM,Gafni RI,Abad V,Baron J.Effects of estrogen on growth plate senescence and epiphyseal fusion.Pro Natl Acad Sci U S A.2001;98:6871-6876.
26.Gruber HE,Ingram JA.Basic Staining Techniques for Cartilage Sections.In:An YH,Martin KL,eds.Handbook of histology methods for bone and cartilage.Totowa,NJ:Humana Press;2003:287-293.
27.Dobie R,Ahmed SF,Staines KA,et al.Increased linear bone growth by GH in the absence of SOCS2 is independent of IGF-1.J Cell Physiol.2015;230:2796-2806.
28.Ballock RT,O'Keefe RJ.Physiology and pathophysiology of the growth plate.Birth Defects Res C Embryo Today.2003;69:123-143.
29.Chung UI.Essential role of hypertrophic chondrocytes in endochonral ossification in bone development.Endocr J.2004;51:19-24.
30.Mackie EJ,Ahmed YA,Tatarczuch L,Chen KS,Mirams M.Endochondral ossification:how cartilage is converted into bone in the developing skeleton.Int J Biochem Cell Biol.2008;40:46-62.
31.Rolian C.Developmental basis of limb length in rodents:evidence for multiple divisions of labor in mechanisms of endochondral bone growth.Evol Dev.2008;10:15-28.
32.Walzer SM,Cetin E,Grubl-Barabas R,et al.Vascularization of primary and secondary ossification centers in the human growth plate.BMC Dev Biol.2014;14:36.
33.Enishi T,Yukata K,Takahashi M,Sato R,Sairyo K,Yasui N.Hypertrophic chondrocytes in the rabbit growth plate can proliferate and differentiate into osteogenic cells when capillary invasion is interposed by a membrane filter.PLoS ONE.2014;9:e104638.
34.Kronenberg HM.Developmental regulation of the growth plate.Nature.2003;423:332-336.
35.Yang L,Tsang KY,Tang HC,Chan D,Cheah KS.Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation.Proc Natl Acad Sci U S A.2014;111:12097-12102.
36.de Crombrugghe B,Lefebvre V,Nakashima K.Regulatory mechanisms in the pathways of cartilage and bone formation.Cur Opin Cell Biol.2001;13:721-727.
37.Farnum CE,Lee R,O'Hara K,Urban JP.Volume increase in growth plate chondrocytes during hypertrophy:the contribution of organic osmolytes.Bone.2002;30:574-581.
38.von Pfeil DJ,DeCamp CE.The epiphyseal plate:physiology,anatomy and trauma.Compend Contin Educ Vet.2009;31:E1-E11.
39.Nilsson O,Baron J.Fundamental limits on longitudinalbone growth:growth plate senescenceand epiphyseal fusion.Trends Endocrinol Metab.2004;15:371-374.
40.Cooper KL,Oh S,Sung Y,Dasari RR,Kirschner MW,Tabin CJ.Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions.Nature.2013;495:375-378.
41.Mohammad KS,Chirgwin JM,Guise TA.Assessing new bone formation in neonatal calvarial organ culture.Methods Mol Biol.2008;455:37-50.
42.Martin EA,Ritman EL,Turner RT.Time course of epiphyseal growth plate fusion in rat tibiae.Bone.2003;32:261-267.
43.Nilsson O,Marino R,De Luca F,Phillip M,Baron J.Endocrine regulation of the growth plate.Horm Res.2005;64:157-165.
44.Shim KS.Pubertal growth and epiphyseal fusion.Ann Pediatr Endocrinol Metab.2015;20:8-12.
45.Wilsman NJ,Farnum CE,Green EM,Lieferman EM,Clayton MK.Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates.J Orthop Res.1996;14:562-572.
46.Gkiatas L,Lykissas M,Kostas-Agnantis L,Korompilias A,Batistatou A,Beris A.Factors affecting bone growth.Am J Orthop(Belle Mead NJ).2015;44:61-67.
47.Locatelli V,Bianchi VE.Effect of GH/IGF-1 on bone metabolism and osteoporsosis.Int J Endocrinol.2014;2014:235060.
48.Giustina A,Mazziotti G,Canalis E.Growth hormone,insulin-Like growth factors,and the skeleton.Endocr Rev.2008;29:535-559.
49.Williams JL,Do PD,Eick JD,Schmidt TL.Tensile properties of the physis vary with anatomic location,thickness,strain rate and age.JOrthop Res.2001;19:1043-1048.
50.Mangiavini L,Merceron C,Schipani E.Analysis of mouse growth plate development.Curr Protoc Mouse Biol.2017;6:67-130.
51.Salic A,Mitchison TJ.A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc Natl Acad Sci U S A.2008;105:2415-2420.
52.Srinivas V,Shapiro IM.The Epiphyseal Growth Plate:the Engine That Drives Bone Elongation.In:Preedy VR,ed.Handbook of growth and growth monitoring in health and disease.New York,NY:Springer;2012:1331-1349.
53.Villemure I,Stokes IA.Growth plate mechanics and mechanobiology.A survey of present understanding.J Biomech.2009;42:1793-1803.