Effect of prostaglandin E1 on regional haversian remodeling in beagles with fractured ribs: A histomorphometric study

Bone, 8, 87-90 (1987) Printed in the USA. All rights reserved.

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8756-3282187 $3.00 + .OO 0 1987 Pergamon Journals Ltd.

Effect of Prostaglandin E, on Regional Haversian Remodeling in Beagles with Fractured Ribs: A Histomorphometric Study M.-S. SHIH and R.VV. NORRDIN Department of Pathology, College of Veterinary Medicine Fort Co//ins, Colorado, U.S.A.

& Biomedical

Sciences,

Colorado

State University,

Address for correspondence and reprints: M.S. Shih, D.V.M., Ph.D Department of Pathology, Health Sciences Center, University of Western Ontario, London, Ontario N6A 321, Canada.

Abstract

formation phases. Osteoclasts and osteoblasts are the major cells involved in resorption and formation, respectively. The processes involved in bone healing are known to include the induction of regional reparative reactions, formation of granulation tissue, and the eventual reconstruction of bone to recover its biomechanical strength. The tunneling of the haversian channels to bridge the fracture ends is histologically evident during bone healing. However, no information about quantitative histomorphologic changes in the haversian envelope adjacent to fracture sites has been reported to date. Frost (1983) proposed that a regional acceleratory phenomenon (RAP) exists at a fracture site. This involves an acceleration of normal ongoing remodeling activity in both hard and soft tissue constituents and enables the injured bone to adapt to a new situation within a short period of time. The RAP appears clinically in the form of an increase in local blood flow and an increase in bone turnover after a sufficient stimulus is applied to the bone (Frost, 1983). Fracture in the present study represents a strong stimulus to bone. Factors involved in initiating the acceleration of remodeling changes after fractures have not yet been determined but could provide clues to activation of remodeling processes in general. During the last decade, prostaglandins (PGs), a family of cyclized derivatives of unsaturated long-chain fatty acids, have been found to be intimately associated with bone metabolism (Raisz and Kream, 1983; Ueno et al., 1985). Among the various PGs studied, the prostaglandins of the E series have been recognized to have significant effects on bone cell-mediated matrix formation and resorption (Raisz and Kream, 1983). In addition, PGE, was found to induce a condition resembling’ infantile cortical hyperostosis during the treatment of patent ductus arteriosus (Ueda et al., 1980). Evidence that PGs may be an important factor in increasing bone mass is also found in the studies of normal, intact experimental subjects (Jee et al., 1985) and in fracture healing (Lund et al., 1982). Dekel et al. (1981) found an increase in PGE concentration at a fracture site. The reestablishment of blood supply to a fracture is very rapid (McKibbin, 1978) and is thought to integrate with bone formation (Paradis et al., 1985; Klein et al., 1985). PGs are known to induce neovascularization in the cornea experimentally (BenEzar, 1978) and are thought to have a role in angiogenesis in the growth of

A histomorphometric study was carried out on healing defects in the ribs of beagles. A transverse fracture was made surgically in the midshaft of the left 9th and 10th ribs. Ten beagles received injections of either a buffer vehicle (n = 4) or prostaglandin E, (PGE,) at a dose of 0.4 mg/day locally (17 = 6) into the fracture sites for a IO-day period and were killed 30 days after the surgery. Double-pulsed fluorescent labels were given with each of two fluochrome markers, calcein before surgical treatment and oxytetracycline hydrochloride before killing. The objectives were to determine manifestations of the regional acceleratory phenomenon (RAP) as changes in regional remodeling in the haversian envelope induced by fracture, effects of PGE, on modification of the RAP in the haversian envelope, and systemic effects of PGE, on remodeling changes of the contralateral matching sites. The differences in haverSian remodeling between the injured and uninjured ribs of the experimental dogs indicated an increase in activation frequency, that is, regional acceleratory phenomenon. The significant effect of PGE, on enhancing fracture-induced acceleration of haversian remodeling was doubtful, because of the preexistent biologic differences found in the two experimental groups. Nevertheless, the two groups shared a similar pattern of remodeling activity. The posttraumatic mineralization that declined at the sampled sites need further investigation. Key Words: Fracture-Healing-Regional Phenomenon-Cortical Bone-PGE,.

Acceleratory

Introduction Mature bone in beagles, as in human beings, contains numerous remodeling sites that react and adjust to biomechanical and biochemical influences. These sites have been referred to as basic multicellular units (BMUs) of bone (Frost, 1969). A remodeling site represents a cycle of successive events occurring at that particular focus, which consists of activation, resorption, resting (or reversal), and a7

88

M.-S. Shih and R.W. Norrdin

tumors (Form et al., 1982). It has been suggested that locally increased PG levels are one of the reactions of bone to trauma and that PG may stimulate differentiation and proliferation of osteoprogenitor cells during bone healing. The objectives of this study were to evaluate by quantitative histomorphometry the regional remodeling response in the haversian envelope after a fracture as well as the modification of this response by locally administered PGE,.

Materials and Methods Ten dogs randomly selected from a beagle colony (Collaborative Radiological Health Laboratory, Colorado State University, Fort Collins, Colorado) were divided into two groups. The randomization was done by computer program (Upjohn) on the basis of age, sex, whether animals are littermates, and clinicopathologic data from a pretreatment profile. The untreated group of dogs contained two 5-7 years and two older than 8 years, and the PGE,treated group of dogs contained two 2-4 years, two 5-7 years, and two older than 8 years. The body weight was 33.20 + 5.13 pounds in the untreated group and 28.28 * 4.63 pounds in the PGE,-treated group. After the dogs were placed under general anesthesia with metafane, fractures at the midshaft of the left 9th and 10th ribs were induced surgically with a pair of bone cutters. Either 0.5 ml 10% ethanol tris buffer vehicle (n = 4) or PGE, (Upjohn Company, Kalamazoo, Michigan) at a dose of 0.5 ml (0.2 mg PGE, in 10% ethanol tris buffer, rt = 6) was injected directly into the two fracture sites twice a day for IO days after surgery. The dosage given was based on preliminary studies conducted at the Upjohn Company (W.B. High, unpublished data). The fractures were allowed to heal without further manipulation. The dogs were killed on the 30th day of the experiment. All dogs received two double pulses of fluochrome labels: (1) calcein (Upjohn) was given at a dose of 15 mgikg per day with a 2-6-2 schedule (2 days on, 6 days off, 2 days on), with the last dose given on the day before the surgery, (2) oxytetracycline hydrochloride (Upjohn) was given at a dose of 25 mglkg per day, with the same schedule, with the last dose given 4 days before killing, The procedure would allow detection of bone mineralization before and 30 days after surgical manipulation, with calcein giving a green color and oxtetracycline giving a yellow color under a UV microscope (Carl Zeiss Ltd., West Germany) with a filter lens (No. 487718, Carl Zeiss Ltd., West Germany). Bone samples were collected at necropsy. They were 8 mmlong transverse sections of the left 9th and 10th ribs taken 2 cm proximal to the fracture sites and matched contralateral segments, Plastic (methyl methacrylate) embedding procedures were used to provide undecalcified bone sections for histomorphometric studies. Sections were cut 100 pm thick from each block on an lsomet slow speed saw (Buehler Ltd., Evanston, Illinois) These sections were hand ground to 40-50 pm in thickness and were stained with toluidine blue (pH 7.2), then evaluated under the microscope (Leitz Wetzlat, West Germany) with an lntegrationsplate I grid (Carl Zeiss, West Germany) and a computerized image-analyzer (Videoplan. Carl Zeiss, West Germany). Using standard histomorphometric parameters and techniques (Reeker, 1983), three consecutive sections were evaluated, comprising at least 50 mm2 of compacta (Frost, 1969). The following primary data on the remodeling parameters of the haversian envelope were collected: (1) total cortical area (mm*), (2) porosity (%), (3) osteoid seam width (pm)s (4) mean wall thickness (km), (5) number of remodeling sites with osteoid seams (no./mm2f, (6) number of remodeling sites with scalloped surfaces (no./mm2), (7) number of remodeling sites with single or double label (no./mm’), and (8) distance between the double labet (pm). Items (1) and (2) were evaluated with the Leitz microscope with the ocular grid (x loo), and the others were carried out with a Zeiss Videoplan with bright or ultraviolet light sources

PGE, effect on RAP In cortical bone after fracture

(x 160). In counting remodeling sites with osteoid seams or sites with scalloped surface, one site would have been counted once in each category if it contained both osteoid and scalloped surfaces. The primary data were converted to secondary data as (1) appositional rate (pm/day). (2) formation rate (mm3/mm2 per year), (3) resorption rate (mm3/mm2 per year), (4) mineralization lag time (days), (5) differences in the number of remodeling sites with labels, in the appositional rate, in the formation rate before and after surgery, (6) formation period (days) and (7) resorption period (days). Item (5) denoted the net change of the parameters before and after the experiment by subtracting the value before surgery from the value 30 days after surgery. One sample (left 10th rib) from one dog in the PGE, group was lost during processing. Thus, the data represent 8 samples of each side in the untreated group, 12 samples of the uninjured side, and 11 samples of the injured side in the PGE,-treated group. The data were analyzed with the Statistical Package for the SocialSciences (SPSS) (Nie et al., 1978). The averaged data from the 9th and the 10th ribs were treated as observations in one population in the statistical analysis. A paired two-tailed f-test was used to compare the averaged group differences between the injured side and the uninjured side in each group. An unpaired two-tailed”t-test was used to compare a parameter in the injured ribs or in the uninjured ribs between the untreated and the PGE,treated groups.

Results Data denoting remodeling condition before surgery showed that the number of labeled remodeling sites before surgery was significantly greater (P < 0.01) in the treated group, but the appositional rate before surgery was slower (P < 0.05) (Table I). In the untreated group, the bone formation rate was significantly faster on the injured side (P < 0.05). The number of remodeling sites with labels at necropsy were decreased from the level before surgery on both injured and uninjured sides, with less decrease on the injured side (P ==I0.01). Other parameters were not significantly different between the two sides. In the PGE,-treated group, the percentage of cortical porosity was significantly greater on the injured side (P < 0.05). The rest of parameters were not significantly different between the two sides. Comparing the injured sides in the PGE,-treated and untreated groups. The percentage of cortical porosity (P < 0.05), the number of remodeling sites with osteoid, and sites with labels (P < 0.01) were greater in the PGE,treated group. The resorption rate was faster (P < 0.01) and the resorption period was shorter (f < 0.05) in the treated group. Comparing the uninjured sides in the PGE,-treated and untreated groups, the significantly different parameters were similar to the comparisons of the injured sides between the two groups. In addition, osteoid seams were significantly.thicker (f < 0.01) in the PGE,-treated group.

Discussion The purposes of the present investigation are partially fulfilled, and the effect of PGE, cannot be assured because of the prevailing biologic differences between the two experimental groups. The numbers of remodeling sites labeled with calcein

89

M.-S. Shih and R.W. Norrdin: PGE, effect on RAP in cortical bone after fracture

Table I. Regional haversian remodeling

adlacent

to rib fracture Untreated

Total cortical area, mm2 Cortical porosity, % Osteoid seam thickness, pm No. of sites with osteoid, no./mm2 No. of sites with scalloped surface, no./mm* No. of sites with labels before SM, no./mm2 No. of sites with labels at necropsy, no./mm* Net change of No. of sites with labels from SM to necropsy, no./mm2 AppositIonal rate before SM, km/day Appositional rate at necropsy, pm/day Net change in appositional rate from SM to necropsy, pmlday Formation rate at necropsy, mmVmmz per year Resorption rate at necropsy, mmYmm* per year Mean wall thickness, km Formation period at necropsy, days Resorption period at necropsy, days Mean total remodeling period, days Mineralization lag time at necropsy, days a Mean (SD). b.c Significant d,e Significant f.QSianificant SM, surgical

Uninjured

Side

10.21 3.03 6.70 4.99 5.31 0.57 0.35

(2.56) (1 53)

-022 1 13 0.96 -0.17 0.03 0.41 68.59 72.96 79.07 152.03 7.11

Group

PGE,-treated

Injured Side

Uninjured

Side

Group Injured Side

(2.51) (1.76) (0.74) (1.71) (1.65j (0.25) (0.26)

9.30 4.18 7.60 12.16 6.16 1.67 1.31

(1.80) (2.02)

(1.81j (0.19) (0.25)

10.51 3.89 7.22 4.27 7.07 0.52 0.48

ii:::; (0.14)

-0.03 1 06 1.15

ii:;:; (0.26)

-036 0 98 0.98

(0.81) (0.09)’ (0.07)

- 0.04 0.93

0.01 0.04 0.79 67.37 68.94 36.35 105.29 7.77

(011)

0.05 0.05 0.81 68.06 69.17 33.37 102.54 7.90

i::;;; (0.28) (3.20) (11.16) (34.59) (0.92)

0.10 0.06 0.26 67.71 61.43 117.66 179.09 6.70

(0 27) (0 02)b ii::;; (11.85) (60 52) (1.84)

(2.08j (1 .02)Q (0.74)Q

;;:,D;; ;::::i’ (14 90)’ (0.51)

8.90 6.02 7.82 14.28 6.80 1.68 1.64

1 00

(1.59) (1 .64)b.d (0.36) i::::;, (1 .Ol) (1.20)e (1.29) (0.09)d (0.08)

;:,::i I!::;‘;” (6.40) (9.94)d (0.61)

difference between uninjured and injured sides of the dogs within the group at P < 0.05.b < O.Ol.c difference on comparison of injured sides in non-treated vs. PG treated group at P < 0.05,d
given before surgery are significantly different between the two groups on both sides. This indicates that there are preexistent biologic differences between these two groups in the bone turnover rate on the haversian envelope. Ap-

parently, age is the factor that caused variations between the two groups before surgical manipulation, since two younger dogs are included in the treated group. Therefore, the significant differences in remodeling parameters between these two groups should be interpretated with caution. The RAP is manifested as an increase in activation of remodeling in favor of osteoid formation adjacent to a fracture healing site (Shih and Norrdin, 1985). The present investigation suggests that similar changes occur in the haversian bone adjacent to the fracture site even with injection of fluid. Global acceleration of remodeling activity in our experimental dogs is suggested based on the data showing higher numbers of remodeling sites per unit of cortical area (mm’) with osteoid (formation) and with scalloped surfaces (resorption) on the uninjured side in the untreated group as compared to other histomorphometric studies in young beagles (Anderson and Danylchuk. 1979; Jorch and Anderson, 1980). It has been postulated that giving sufficient stimulus to a bone can induce RAP in the contralateral side of the body (Frost, 1983). Although comparisons between the two experimental groups cannot be used to verify the effect of PGE, on enhancing RAP induced by fracture, the pattern of remodeling activity in either injured or uninjured ribs of the treated dogs is similar to that in the untreated dogs. This leads to the suggestion that PGE, may be a factor that activates RAP in bone healing. It has been stated that RAP can exist as long as 4 rnonths to 2 years (Frost, 1983), and it may continue to be present until the fracture is completely healed. The activation of new BMUs is assumed to be at a con-

stant rate through the healing period. Therefore, bone remodeling on the haversian envelope adjacent to a fracture site can be considered to be in a steady, high turnover state. This is supported by the previous analysis that no age effect on subjects responding to fractures was noted at the end of a 30-day trial (Shih and Norrdin, 1985). The parameters denoting the net change in number of labeled remodeling sites show a uniform decrease at the end of the experiment from the level before surgery on both sides in both groups. However, the injured sides tend to be less depressed, and this is significant (P < 0.01) in the untreated group. This indicates a mineralization decline at the sampled sites after fracture, which coincides with observations in fracture cases evaluated on radiographs (Anderson and Nilsson, 1979) and considered as a constant in fracture cases (Nilsson and Obrant, 1983). It has been recognized that mineralization of osteoid in vivo occurs continuously after it is laid down on the bone surface until the completion of a BMU-the so-called ON phase (Frost, 1980; Hori et al., 1985; Takahashi et al., 1985). However, interruption of mineralization in different BMUs of the same specimen (OFF phase) has been noted in young, healthy subjects by the same investigators. Age did not seem to interfere with mineralization in fractured dogs in a previous study (Shih and Norrdin, 1985). It is difficult to explain, then, why mineralization defects occur in fracture cases, since it is supposed to participate in reconstruction of the bone defect. There is no hard evidence indicating what mechanism turns mineralization on and off (Frost, 1980). Such factors as parathyroid hormone (PTH), calcitonin (CT), and vitamin D metabolites were considered important during fracture healing (Meller et al., 1984; Lips et al., 1985). The serum level of PTH, CT, and vitamin D metabolites and the urinary excretion rate of calcium and phosphate were not monitored in this study, but serum calcium

90

M.-S. Shih and R.W. Norrdin. PGE, effect on RAP in cortical bone after fracture

and phosphate concentrations were not significantly different. Local factors thought to participate in mineralization (Hori et al., 1985) deserve further investigation. However, the present study does not provide evidence that PGE, is involved in inducing mineralization. In the present study, the hydrostatic pressure applied to the local cells and tissues through direct injection of fluid may have masked the biochemical effects of PGE. Endogenous PG production can be induced by physical stress (Somjen et al., 1980) and high dosage (Jee et al., 1985; Ueno et al., 1985) or high concentration (Raisz and Kream, 1983) can inhibit bone formation and resorption. However, a similar pattern of remodeling activity is noted when the data using oral PGE, (Shih and Norrdin, 1986) and the data using locally injected PGE, are compared. This suggests that local injection of PGE does not obviously interfere with the appearance of remodeling during healing.

We acknowledge the cially Drs. J.C Babcock and W.B. High, assistance in completing these studies. Dr. Colin Anderson at the University of critical reading of the manuscript and typing the manuscript. Acknowledgment:

Upjohn Company, espefor support and financial We are also indebted to Western Ontario for hrs Mrs. Wendy Foster for

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Received Apnl22, 1986 Revised September 2, 1986 Accepted: September 30, 7986