- Email: [email protected]
Regional acceleration of remodeling during healing of bone defects in beagles of various ages
Bone, 6, 377-379 (1985) Prtnted in the USA. All rights reserved
Copyright
8756-3282185 $3.00 + .OO @ 1985 Pergamon Press Ltd.
Regional Acceleration of Remodeling During Healing of Bone Defects in Beagles of Various Ages M.S.
SHIH and R.W. NORRDIN
Department
of Pathology,
College of Vetennary Medicme and B/omed\ca/ Sciences, Colorado
Introduction
State University, Fort Collins, CO, USA
of left tibia taken 2 cm proximal to a drill hole defect In the medral cortex at midshaft. and cross sectrons of left 9th and 10th ribs taken 2 cm proximal to a transverse fracture made with bone cutters. Contralateral matched tibia and rib cross sections were used for comparison. Dogs of three different age groups (2-4 years, n = 2; 5-7 years, n = 4; and 8-11 years, n = 4) were included In the study. Six beagles were orally given capsules contarnmg ethyl alcohol vehicle for 30 days postinjury, and four beagles were locally Injected with a 10% alcohol-Tris buffer vehicle (0.5 ml, brd) in the healing sites for the first 10 days. The dogs received two double pulses of fluochrome labels, the first, calcein 15 mg/kg. before surgery and the second, oxytetracycline 25 mg/kg, before sacrifice wrth a 2-6-2 schedule (2 days dosed, 6 days off, 2 days dosed) on each admrnistratron. The last injectron of the pretreatment labels was given 1 day before surgery, and the last of the posttreatment labels was given 4 days before killing at 30 days Three 100 pm thick sections were cut from each plastic-embedded block with a low-speed bone saw (Isomet). Sections were ground to 40-50 pm thickness and statned lightly with tolurdtne blue (pH 7.2). The same slides were examined with the brightfield and UV microscopes. Primary parameters of the remodeling activrty in the haversian, periosteal, and corticoendosteal envelopes of the tibia and ribs were measured as well as the trabecular surfaces of the ribs. These rncluded osteoblast, osteoid, and osteoclast-covered surface Scalloped surface without osteoclasts was expressed as inactive resorption surface. The measurements of labels (both double and single), fractional surfaces, volume of periosteal prolrferative bone, and distance between double labels was performed usrng a computerized Image analysis system (Zeiss Videoplan), whereas other parameters, such as percent porosity, cortrcal area, and trabecular volume, were measured on a Leitz mrcroscope wrth an ocular grid (Integrations plate II). When bone proliferatron was present on the periosteal or corticoendosteal surface, we considered as surface length an imaginary line along the base of the proliferation. The data were entered on a computer program for statistical analysis The Student two-tailed t test was used to compare the mean values for each parameter of remodelrng on the healing and control side. Analysis of vanance was used to look for age or local treatment effects on each parameter
In the course of studies on bone healing processes (Voigt et al., 1985; Collier et al., 1985), it was observed that striking changes occur in remodeling adjacent to the site of injury. This phenomenon, which represents part of the reaction to injury and mobilization of resources to bring about stabilization and repair, usually involves an acceleration of normal activttres in both hard and soft tissues and has been described as the “regronal acceleratory phenomenon” (RAP) by Frost (1983). There is evidence that this can be affected by systemic factors in the increased response to periosteal stripping In experimentally hyperthyroid dogs (High et al., 1981). The factors involved in the initiation of the remodeling changes have not been studied but could provide clues to the activation of remodeling processes in general. In the present study, drill hole defects in the tibia and nb fracture sates were used to provide models of healing bone. At the sites themselves there was resorption of devitalized bone at the injured edges and new bone formed. Remodeling sttes In the Immediately adjacent bone were Irregularly oriented with respect to the long axis of the bone and had frequent branching. These contained newly formed vascular channels and emphasized the intimate relation between remodeling and vascularization of the healing site. Further back from the margin of injury, the changes were not nearly so dramatic or irregular, and remodeling changes appeared to blend with ongotng processes. Increased haversian remodeling sites, for example, appeared to be orderly extensons of preexisting longitudinally orrented haversian systems. It was from thts region, 2 cm from the injury, that regtonal changes in remodelrng were studied. The objective was to quantitatively evaluate the regional remodeling response by comparison with the contralateral uninjured bone. In additron, some dogs had received local injectrons of an alcohol-Tris buffer vehicle in the healing sites. The dogs used in the study varied from 2 to 11 years of age, and the data were evaluated for an effect of age on the regional remodeling changes.
Results The significant changes in remodeling parameters in the tibia are presented in Table I with the level of statistical probability. In the periosteum, bone proliferation was present on the healing side at this cross-sectional level in 3 of the 10 dogs, and the mean proportion of surface with posttreatment label was Increased. In the haversian bone the porosity and number of sites labeled per mm2 were increased on the healing side. In the corticoendosteum, the remodeling ac-
Materials and Methods Bone samples were collected at necropsy from 10 beagles, which represented the control groups in a study of the effects of orally and locally admrnrstered prostaglandins on bone healrng at 30 days (Vorgt et al., 1985). The samples were 8 mm thrck cross sectrons 377
378
4th Inlernatjonal
Bone Hlstomorphometry
Workshop
Table 1. Regional remodeling changes during healing in the tibia.
Periosteal Single pretreatment
label, %
Single posttreatment label, % Bone proliferation, no./group Haversian Cortical porosity, % Posttreatment labeled sites, no./mm2 Cortical-Endosteal Osteoid surface, % Osteoblast surface, % Osteoclast surface, % Single posttreatment label, % Double posttreatment label, % Bone proliferation. no./group
Control side
Healing side
Probablllty
3 11 (2.60)la 0.60 (0.13) o/10
4.32 (6.04) 22.07 (18.31) 3/10
0.019 0.000
2.29 (0.90) 0.11 (0.08)
4.05 (1.92) 1.37 (1.39)
0.034 0.000
15.56 (8.87) 0.10 (0.21) 2.23 (1 14) 1.80 (2 21) 0.19 (0.38) o/10
31.94 (18.65) 1.58 (2.44) 2.91 (2.41) 37.12 (19.99) 13.02 (13.94) 8110
0.037 0.000 0.037 0.000 0 000
‘* Standard deviation.
on the healing side were clearly stimulated with greater increases in formation than resorption. The proportion of osteoid surface with plump osteoblasts was low relative to osteoid surface. Often the osteoblasts were elongated and the matrix of a more woven type. The number of dogs with bone proliferation on the corticoendosteal surface was greater than on the periosteal surface. The changes in the ribs are listed in Table II. The scalloped resorption surface and posttreatment fluochrome labeling were increased on the periosteal surface on the healing side. No gross bone proliferation was seen, however. The cortical porosity and number of labeled remodeling sites had higher mean values on this side but were not significantly different. On the corticoendosteal envelope the formatlon parameters of osteoid surface and posttreatment fluochrome labeling are decreased on the healing side. The mean trabecular volume was less but not significantly dlfferent on the healing side. Scalloped resorption surface was increased, and osteoblast and double-labeled surface was decreased, which would lead to a negative bone balance on this side. Single posttreatment label was greater, however, suggesting that the amount of osteoid mineralizing tivities
Table
II. Regional remodeling
Discussion In this study we evaluated regional remodeling changes during healing in dogs subjected to a standardized injury 30 days earlier. In the tibia, penosteal bone formation was in-
changes during healing in the nbs
Periosteal Double pretreatment label, % Single posttreatment label, % Double posttreatment label, % lnactlve resorption surface,% Haversian Cortical porosity, % Posttreatment labeled sites, no./mm2 Corticoendosteal Osteold surface, % Double pretreatment label, % Single posttreatment label, % Trabecular Trabecular bone volume, % Osteoblast surface, % Inactive resorption surface, .% Single pretreatment label, % Single posttreatment label, % Double posttreatment label, % a Standard dewatlon
between the first and second labels was decreasing. Compared to pretreatment labels, trabecular formation surface was decreasing in both healing and control side. The significant differences seen in pretreatment labels in the tibia1 periosteum (higher on the healing side) and the rib periosteum, corticoendosteum, and trabecular surfaces (higher on the control side) are difficult to explain. They suggest significant right-left differences prior to surgery. It is possible that some of these labels were resorbed following surgery. Analysis of variance of the pooled data for remodeling changes revealed that there was no age effect in any of the parameters of posttreatment response. There was, however, a significantly greater number of labeled cortical remodeling sites in the dogs given local injections for the first 10 days.
Control side
Healing side
142 1.29 0.17 3.93
0 81 6.29 0.06 6.36
(2.75)” (2.98) (0 51) (2 32)
3.12 (1.85) 0 52 (0.49) 1369 (699) 4.90 (4.48) 441 (328) 14 87 (6 64) 0 93 (0.90) 1046(243) 20.84 (7 62) 7.80 (5.02) 0.89 (1.31)
(1 18) (9.28) (0.27) (4.22)
Probablllty
0 0 0 0
3 69 (1 53) 0.76 (0 66) 8.55 (12 52) 2 06 (2 23) 3 92 (8 90) 12 25 0.67 14 43 13 87 963 0 29
(4 01) (0 45) (5 10) (4 17) (1266) (0 42)
001 000 008 012 NS NS
0 015 0.004 0 000
0 0 0 0 0
NS 004 002 012 000 000
4th International
Bone
Histomorphometry
379
Workshop
creased, the number of haversian remodeling sites was markedly increased, and there was activation of remodeling and increased bone formation on the corticoendosteal surface. In the ribs, periosteal bone formation was increased, formation on the corticoendosteal surface was decreased, and there was an increase in resorption surface with a declining extent of forming surface in trabecular bone. With two to four dogs in each of three different age groups, no age effect was seen in the response to injury. The effect of local injection was to increase the number of haversian remodeling sites with posttreatment fluochrome labels. The data were characterized by marked individual variation. In the rib this may have been due to the variable amount of overriding and instability in individual fracture sites. In the tibia, although there was greater stability, the partial immobilization associated with limping and a variable degree of damage to the underlying marrow tissue (e.g., vasculature) in making the cortical drill hole defect may be influencing factors. In terms of the relationship of the regional remodeling changes to healing of the defects, it is interesting to note that healing of the tibia1 defect was slower in the dogs that had received local injections of the alcohol-Tris buffer vehicle (Voigt et al., 1985), and it was in these dogs that there
was greater haversian remodeling in the present study. This suggests that additional disturbance at the injury site may cause a greater regional acceleration of remodeling and that a greater RAP is associated with slower healing. In this study, the regional changes in remodeling reflected a complex response involving all envelopes of the bone in a variable pattern of change. These changes appeared to represent circulatory adjustments associated with healing and biomechanically induced modeling changes as well as changes in the ongoing processes of remodeling.
References Collrer M.A., Brighton C.T.. Norrdin R.W.. Twardock A.R and Rendano J T.: Drrect current strmulation of bone production In the horse Prelrmrnary study with a “gap healtng” model. Am. J. Vef. Res 46 610-621. 1985. frost H.M.: The regronal acceleratory phenomenon-A rewew Henry ford Hosp Med. J 31:3-g, 1983. Hugh W.B.. Capen CC and Black H.E.. Effects of thyroxtne on corttcal bone remodelrng In adult dogs A hrstomorphometnc study Am J fafhoi. 102:438-446, 1981. Vorgt G.L.. Norrdrn R.W. and Hugh W B: Effects of orally and locally admrnrstered prostaglandrns E, or E,on bone healing In beagles In preparatron, 1985.
Copyright
8756-3282185 $3.00 + .OO @ 1985 Pergamon Press Ltd.
Regional Acceleration of Remodeling During Healing of Bone Defects in Beagles of Various Ages M.S.
SHIH and R.W. NORRDIN
Department
of Pathology,
College of Vetennary Medicme and B/omed\ca/ Sciences, Colorado
Introduction
State University, Fort Collins, CO, USA
of left tibia taken 2 cm proximal to a drill hole defect In the medral cortex at midshaft. and cross sectrons of left 9th and 10th ribs taken 2 cm proximal to a transverse fracture made with bone cutters. Contralateral matched tibia and rib cross sections were used for comparison. Dogs of three different age groups (2-4 years, n = 2; 5-7 years, n = 4; and 8-11 years, n = 4) were included In the study. Six beagles were orally given capsules contarnmg ethyl alcohol vehicle for 30 days postinjury, and four beagles were locally Injected with a 10% alcohol-Tris buffer vehicle (0.5 ml, brd) in the healing sites for the first 10 days. The dogs received two double pulses of fluochrome labels, the first, calcein 15 mg/kg. before surgery and the second, oxytetracycline 25 mg/kg, before sacrifice wrth a 2-6-2 schedule (2 days dosed, 6 days off, 2 days dosed) on each admrnistratron. The last injectron of the pretreatment labels was given 1 day before surgery, and the last of the posttreatment labels was given 4 days before killing at 30 days Three 100 pm thick sections were cut from each plastic-embedded block with a low-speed bone saw (Isomet). Sections were ground to 40-50 pm thickness and statned lightly with tolurdtne blue (pH 7.2). The same slides were examined with the brightfield and UV microscopes. Primary parameters of the remodeling activrty in the haversian, periosteal, and corticoendosteal envelopes of the tibia and ribs were measured as well as the trabecular surfaces of the ribs. These rncluded osteoblast, osteoid, and osteoclast-covered surface Scalloped surface without osteoclasts was expressed as inactive resorption surface. The measurements of labels (both double and single), fractional surfaces, volume of periosteal prolrferative bone, and distance between double labels was performed usrng a computerized Image analysis system (Zeiss Videoplan), whereas other parameters, such as percent porosity, cortrcal area, and trabecular volume, were measured on a Leitz mrcroscope wrth an ocular grid (Integrations plate II). When bone proliferatron was present on the periosteal or corticoendosteal surface, we considered as surface length an imaginary line along the base of the proliferation. The data were entered on a computer program for statistical analysis The Student two-tailed t test was used to compare the mean values for each parameter of remodelrng on the healing and control side. Analysis of vanance was used to look for age or local treatment effects on each parameter
In the course of studies on bone healing processes (Voigt et al., 1985; Collier et al., 1985), it was observed that striking changes occur in remodeling adjacent to the site of injury. This phenomenon, which represents part of the reaction to injury and mobilization of resources to bring about stabilization and repair, usually involves an acceleration of normal activttres in both hard and soft tissues and has been described as the “regronal acceleratory phenomenon” (RAP) by Frost (1983). There is evidence that this can be affected by systemic factors in the increased response to periosteal stripping In experimentally hyperthyroid dogs (High et al., 1981). The factors involved in the initiation of the remodeling changes have not been studied but could provide clues to the activation of remodeling processes in general. In the present study, drill hole defects in the tibia and nb fracture sates were used to provide models of healing bone. At the sites themselves there was resorption of devitalized bone at the injured edges and new bone formed. Remodeling sttes In the Immediately adjacent bone were Irregularly oriented with respect to the long axis of the bone and had frequent branching. These contained newly formed vascular channels and emphasized the intimate relation between remodeling and vascularization of the healing site. Further back from the margin of injury, the changes were not nearly so dramatic or irregular, and remodeling changes appeared to blend with ongotng processes. Increased haversian remodeling sites, for example, appeared to be orderly extensons of preexisting longitudinally orrented haversian systems. It was from thts region, 2 cm from the injury, that regtonal changes in remodelrng were studied. The objective was to quantitatively evaluate the regional remodeling response by comparison with the contralateral uninjured bone. In additron, some dogs had received local injectrons of an alcohol-Tris buffer vehicle in the healing sites. The dogs used in the study varied from 2 to 11 years of age, and the data were evaluated for an effect of age on the regional remodeling changes.
Results The significant changes in remodeling parameters in the tibia are presented in Table I with the level of statistical probability. In the periosteum, bone proliferation was present on the healing side at this cross-sectional level in 3 of the 10 dogs, and the mean proportion of surface with posttreatment label was Increased. In the haversian bone the porosity and number of sites labeled per mm2 were increased on the healing side. In the corticoendosteum, the remodeling ac-
Materials and Methods Bone samples were collected at necropsy from 10 beagles, which represented the control groups in a study of the effects of orally and locally admrnrstered prostaglandins on bone healrng at 30 days (Vorgt et al., 1985). The samples were 8 mm thrck cross sectrons 377
378
4th Inlernatjonal
Bone Hlstomorphometry
Workshop
Table 1. Regional remodeling changes during healing in the tibia.
Periosteal Single pretreatment
label, %
Single posttreatment label, % Bone proliferation, no./group Haversian Cortical porosity, % Posttreatment labeled sites, no./mm2 Cortical-Endosteal Osteoid surface, % Osteoblast surface, % Osteoclast surface, % Single posttreatment label, % Double posttreatment label, % Bone proliferation. no./group
Control side
Healing side
Probablllty
3 11 (2.60)la 0.60 (0.13) o/10
4.32 (6.04) 22.07 (18.31) 3/10
0.019 0.000
2.29 (0.90) 0.11 (0.08)
4.05 (1.92) 1.37 (1.39)
0.034 0.000
15.56 (8.87) 0.10 (0.21) 2.23 (1 14) 1.80 (2 21) 0.19 (0.38) o/10
31.94 (18.65) 1.58 (2.44) 2.91 (2.41) 37.12 (19.99) 13.02 (13.94) 8110
0.037 0.000 0.037 0.000 0 000
‘* Standard deviation.
on the healing side were clearly stimulated with greater increases in formation than resorption. The proportion of osteoid surface with plump osteoblasts was low relative to osteoid surface. Often the osteoblasts were elongated and the matrix of a more woven type. The number of dogs with bone proliferation on the corticoendosteal surface was greater than on the periosteal surface. The changes in the ribs are listed in Table II. The scalloped resorption surface and posttreatment fluochrome labeling were increased on the periosteal surface on the healing side. No gross bone proliferation was seen, however. The cortical porosity and number of labeled remodeling sites had higher mean values on this side but were not significantly different. On the corticoendosteal envelope the formatlon parameters of osteoid surface and posttreatment fluochrome labeling are decreased on the healing side. The mean trabecular volume was less but not significantly dlfferent on the healing side. Scalloped resorption surface was increased, and osteoblast and double-labeled surface was decreased, which would lead to a negative bone balance on this side. Single posttreatment label was greater, however, suggesting that the amount of osteoid mineralizing tivities
Table
II. Regional remodeling
Discussion In this study we evaluated regional remodeling changes during healing in dogs subjected to a standardized injury 30 days earlier. In the tibia, penosteal bone formation was in-
changes during healing in the nbs
Periosteal Double pretreatment label, % Single posttreatment label, % Double posttreatment label, % lnactlve resorption surface,% Haversian Cortical porosity, % Posttreatment labeled sites, no./mm2 Corticoendosteal Osteold surface, % Double pretreatment label, % Single posttreatment label, % Trabecular Trabecular bone volume, % Osteoblast surface, % Inactive resorption surface, .% Single pretreatment label, % Single posttreatment label, % Double posttreatment label, % a Standard dewatlon
between the first and second labels was decreasing. Compared to pretreatment labels, trabecular formation surface was decreasing in both healing and control side. The significant differences seen in pretreatment labels in the tibia1 periosteum (higher on the healing side) and the rib periosteum, corticoendosteum, and trabecular surfaces (higher on the control side) are difficult to explain. They suggest significant right-left differences prior to surgery. It is possible that some of these labels were resorbed following surgery. Analysis of variance of the pooled data for remodeling changes revealed that there was no age effect in any of the parameters of posttreatment response. There was, however, a significantly greater number of labeled cortical remodeling sites in the dogs given local injections for the first 10 days.
Control side
Healing side
142 1.29 0.17 3.93
0 81 6.29 0.06 6.36
(2.75)” (2.98) (0 51) (2 32)
3.12 (1.85) 0 52 (0.49) 1369 (699) 4.90 (4.48) 441 (328) 14 87 (6 64) 0 93 (0.90) 1046(243) 20.84 (7 62) 7.80 (5.02) 0.89 (1.31)
(1 18) (9.28) (0.27) (4.22)
Probablllty
0 0 0 0
3 69 (1 53) 0.76 (0 66) 8.55 (12 52) 2 06 (2 23) 3 92 (8 90) 12 25 0.67 14 43 13 87 963 0 29
(4 01) (0 45) (5 10) (4 17) (1266) (0 42)
001 000 008 012 NS NS
0 015 0.004 0 000
0 0 0 0 0
NS 004 002 012 000 000
4th International
Bone
Histomorphometry
379
Workshop
creased, the number of haversian remodeling sites was markedly increased, and there was activation of remodeling and increased bone formation on the corticoendosteal surface. In the ribs, periosteal bone formation was increased, formation on the corticoendosteal surface was decreased, and there was an increase in resorption surface with a declining extent of forming surface in trabecular bone. With two to four dogs in each of three different age groups, no age effect was seen in the response to injury. The effect of local injection was to increase the number of haversian remodeling sites with posttreatment fluochrome labels. The data were characterized by marked individual variation. In the rib this may have been due to the variable amount of overriding and instability in individual fracture sites. In the tibia, although there was greater stability, the partial immobilization associated with limping and a variable degree of damage to the underlying marrow tissue (e.g., vasculature) in making the cortical drill hole defect may be influencing factors. In terms of the relationship of the regional remodeling changes to healing of the defects, it is interesting to note that healing of the tibia1 defect was slower in the dogs that had received local injections of the alcohol-Tris buffer vehicle (Voigt et al., 1985), and it was in these dogs that there
was greater haversian remodeling in the present study. This suggests that additional disturbance at the injury site may cause a greater regional acceleration of remodeling and that a greater RAP is associated with slower healing. In this study, the regional changes in remodeling reflected a complex response involving all envelopes of the bone in a variable pattern of change. These changes appeared to represent circulatory adjustments associated with healing and biomechanically induced modeling changes as well as changes in the ongoing processes of remodeling.
References Collrer M.A., Brighton C.T.. Norrdin R.W.. Twardock A.R and Rendano J T.: Drrect current strmulation of bone production In the horse Prelrmrnary study with a “gap healtng” model. Am. J. Vef. Res 46 610-621. 1985. frost H.M.: The regronal acceleratory phenomenon-A rewew Henry ford Hosp Med. J 31:3-g, 1983. Hugh W.B.. Capen CC and Black H.E.. Effects of thyroxtne on corttcal bone remodelrng In adult dogs A hrstomorphometnc study Am J fafhoi. 102:438-446, 1981. Vorgt G.L.. Norrdrn R.W. and Hugh W B: Effects of orally and locally admrnrstered prostaglandrns E, or E,on bone healing In beagles In preparatron, 1985.