High-Turnover Periprosthetic Bone Remodeling and Immature Bone Formation Around Loose Cemented Total Hip Joints

Authors


Abstract

Aseptic loosening and periprosthetic osteolysis are the major problems awaiting solution in total hip surgery. The clinical investigation focused on the analysis of periprosthetic bone remodeling to clarify one important key event in the cascade of periprosthetic connective tissue weakening and osteolysis around loose artificial hip joints. Twelve acetabular bone samples adjacent to granulomatous synovial-like membrane of loose hip prosthesis were retrieved at revision surgery and processed for Villanueva bone staining for morphological observation and bone histomorphometric analysis. Eight well-fixed bony samples were used as control. Although osteoclastic surface and eroded surface by osteoclasts were evident in the periprosthetic bone from loose hip joints (p = 0.003 and p = 0.027), increased osteoid/low-mineralized bone matrix (p < 0.001) and osteoid width (p < 0.001) also were significant findings in structural analysis. In addition, not only elevated mineral apposition rate (MAR; p = 0.044) but also increased mineralizing surface (p = 0.044) and bone formation rate (BFR; p = 0.002) in loose periprosthetic bones were shown in dynamic data analysis. These results were confirmed by precise morphological observation by confocal laser scanning microscopy. Active coupling of bone formation and resorption and increased osteocytes with abundant bone canalicular projections were found in combined with the presence of immature bone matrices (osteoid and low-mineralized bone areas) in periprosthetic bones from loose hip joints. These results indicated that active osteoclastic bone resorption and/or defective bone formation are coupled with monocyte/macrophage-mediated foreign body-type granuloma in the synovial-like interface membrane of loose hip joints. Thus, this unique high-turnover periprosthetic bone remodeling with bad bone quality probably is caused by the result of cellular host response combined with inappropriate cyclic mechanical loading. The fragile periprosthetic bone may contribute to hip prosthesis loosening.

INTRODUCTION

TOTAL HIP arthroplasty has become an efficacious and cost-effective procedure in the treatment of patients with painful end-stage arthritis since the concept of low-friction artificial joints was established.(1) The arthroplasty with modern technique makes it possible to relieve pain and restore the daily activity of life in walking. Some 800,000 hip joints are replaced on an annual basis on a worldwide scale. However, approximately 90% of a 10-year survival rate of total hip arthroplasty has been shown, and some of them have to be revised because of aseptic loosening and periprosthetic osteolysis even after insertion of the prostheses technically is well performed.(2,3) This major problem has been a subject of debate for three decades, and there is increasing concern regarding biocompatibility of total hip prostheses.(2–5)

It seems evident that the periprosthetic osteolysis and loosening of technically well-inserted prostheses in part are attributable to cellular host responses in periprosthetic connective tissues.(6–10) Extracellular matrix degradation and connective tissue remodeling around artificial hip joint implants have been considered as major biological events in the process of osteolysis and loss of prosthetic tissue support.(11) The reports by Goldring et al.(6,7) have directed research to biochemical and molecular biological analysis of interfacial synovial-like membrane between bone and implants.(11–23) Intensive research has contributed to clarification of phenomena related to osteolysis and periprosthetic connective tissue weakening as a result of biological host response to implants.

However, despite intensive research, the precise biological mechanisms responsible for loosening have not been clarified completely yet. Particularly, little is known regarding periprosthetic bone remodeling and bone quality around loose implants. This study focused on the investigation of characteristics of periprosthetic bone remodeling to clarify one important key event in the cascade leading to loss of implant support. Bone histomorphometric analysis combined with a confocal laser scanning microscopy system was applied.(24,25)

MATERIALS AND METHODS

Patients and sample preparation

Twelve samples of periprosthetic bone around cemented loose hip prostheses with roentgenographically detectable osteolysis were obtained at revision surgery at Yamagata University Hospital and Saiseikai Yamagata Hospital. The primary surgeries were performed because of primary osteoarthritis in 2 cases and osteoarthritis due to developmental dysplasia in 10 cases; 10 were female and 2 were male, and the mean age was 69.0 years (range, 61–78 years; Table 1). Eight acetabular bony tissues adjacent to well-fixed implants without roentgenographic osteolysis, but revised because of acetabular socket-femoral head component damage, were used as controls (mean age, 67.8 years; range, 62-76 years; six were female and two were male, primary surgeries were performed because of primary osteoarthritis in three cases and dysplastic osteoarthritis in five cases; Table 1). Patients with a history of systemic inflammatory diseases and disorders of bone metabolism were excluded. Serum levels of calcium, phosphate, alkaline phosphatase, blood urea nitrogen, and creatinine of all the patients were within normal limits. Before revision surgery, each patient received in vivo tetracycline labeling as follows: oral tetracycline hydrochloride (250 mg four times daily, Achromycin V; Wyeth-Lederle, Tokyo, Japan) for 2 days followed by a 14-day drug-free interval, and then 2 days of oral tetracycline hydrochloride again (250 mg four times daily). Five days after the end of second labeling, the bony samples, 7 mm × 7 mm, were obtained at revision surgery. The site of sampling was adjacent to interfacial granulomatous membrane between acetabular bone and implants in loose hips and to thin fibrous tissues between acetabular bone and implants in unloose implants. The samples were fixed with 70% ethanol at room temperature for 1 day, followed by immersion in Villanueva bone stain solution (Maruto, Tokyo, Japan)(26,27) for 3 days. The samples were dehydrated through gradient ethanols and embedded in methyl methacrylate resin (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After polymerization, 50-μm sections from bone-implant interface were ground using a Speed Lap Grinder (ML-150DC; Maruto). The study protocol was approved by the Institutional Review Board, Yamagata University, and Saiseikai Yamagata Hospital before initiation and the recommendations of the Helsinki Declaration were followed.

Table Table 1.. List of the Patients with Loose and Unloosened Cemented Hip Joints
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Morphological observations by confocal laser scanning microscopy and bone histomorphometric analysis

Confocal laser scanning microscopy system (TCS SP; Leica Microsystems, Leica, Germany) was used in this study. In the analysis of confocal laser scanning microscopy, the excitation wavelength was set at 488 nm using an acoustic optical tunable filter system. Tetracyclin fluorescence was detected through 515–550 nm, and the autofluorescence of the Villanueva bone stain was detected through 590-650 nm using a spectral confocal microscope. The spectral confocal microscope detected fluorescence as follows: fluorescence was separated to colors by a prism and then the colors were selected with the slits controlling both position and width of wavelength, which were in front of a photomultipliers detector.

Mature-mineralized bone was observed as green-staining structures and low-mineralized bone was observed as orange-staining structures containing osteocytes. Osteoid, counted as both a surface and a volume feature, was observed as red-staining tissue of at least 4-μm thickness at the bone-bone marrow interface. Immature bone was defined as low-mineralized bone and osteoid area. In addition, differential interference constant image transmitted light in the microscopy system was applied to evaluate whether the bone that is formed next implant was woven and/or lamellar. Eroded surface was defined as bone marrow interface with a scalloped or ragged appearance. The surface generally was near osteoid and sometimes lay beneath osteoclasts. Quiescent surface was defined as all surfaces other than osteoid or eroded. Osteoblasts were defined as bone cells directly apposed to osteoid. Osteocytes were defined as bone cells in the mineralized bone matrix with cytoplasmic projection into bone canaliculi. Osteoclasts were defined as multinucleated cells lying within 10 μm of an eroded surface.(26–28) Ten random fields (magnification ×250) from each sample were used to perform histomorphometric analysis, including counting of osteocytes and its density (N.Ot, cell/mm2), by the National Institutes of Health (NIH) image public domain software. Each image was analyzed to determine the periprosthetic bone volume (BV/TV, %) and void volume (Vd.V/TV, %), with the total volume (TV) being comprised of all three phases. Mature-mineralized bone volume (M-Md.V/BV, %), low-mineralized bone volume (L-Md.V/BV, %), osteoid volume (OV/BV, %), and osteoid thickness (O.Th, μm) also were calculated as structural data. Osteoid surface (OS/BS, %), osteoblast surface (Ob.S/BS, %), eroded surface (ES/BS, %), and osteoclast surface (Oc.S/BS) were calculated with each corresponding to perimeter divided by bone surface (BS), that is, as surface data.(28,29) As for the observation of tetracycline labeling, it was detected as yellow or green-yellow color lines or deposition in the calcified matrix, where mineralized bone matrix was detected as dark green or black to enhance the labeling in the confocal laser system. Mineral apposition rate (MAR, μm/day) was calculated by the distances between the midpoints of the two labeled lines, divided by the time between the midpoints of the labeling periods,(30) and mineralizing surface (MS/BS, %) was calculated using the double-labeled surface, plus one-half of the single labeled surface, which was divided by total bone surface. They were used as basic dynamic data.(28,29) Mineralization lag time (Mlt, day) was calculated as mean time interval between deposition and mineralization of any infinitesimal volume of matrix, given by osteoid thickness divided by adjusted apposition rate (O.Th./Aj.AR.; Aj.AR. was given by MARMS/OS). Bone formation rate (BFR) was calculated both by surface referent value (BFR/BS, mm3/mm2 per year) and bone volume referent value (BFR/BV, %/year). They were used as basic and derived dynamic data.(28,29)

Statistics

The mean and SD were calculated, and loose and unloose groups were compared by unpaired t-test for statistical significance. The level of significance was set at p < 0.05.

RESULTS

Morphological observation by confocal laser scanning microscopy

Periprosthetic bones adjacent to loose interface:

In the bony samples adjacent to loose interface, granulomatous connective tissue, including monocytes/macrophages, fibroblasts, and vascular endothelial cells, was observed adjacent to retrieved bone. Infiltration of granulomatous cell components into the periprosthetic bone also was found. Increase of tetracycline-labeled bone surface, osteoid and low-mineralized area, and active osteoblast lining were the significant findings in the periprosthetic bone adjacent to loose interface (Fig. 1A), when compared with unloose interface (Fig. 1B), although osteoclasts, resorption lacunae, and eroded surface also were observed abundantly (Fig. 2), in addition to increased immature bone (osteoid and low-mineralized areas) in the periphery of bone matrices, which were recognized as red staining (osteoid) and orange staining (low-mineralized area), respectively. Scattered low-mineralized areas, which were stained as orange, also were found in the green-stained mature-mineralized bone matrices ( Figs. 1A). The presence of lamellar structure was observed in the periprosthetic bone adjacent to interface tissue, but it was irregular, mosaic, and sometimes intervened by and/or coupled with woven bones and was identified as the area of no lamellar structure (Fig. 3A). Osteocytes, which had abundant dendritic cytoplasmic projections in bone canaliculi, were frequently observed in the low-mineralized areas in the periphery of bone matrices and scattered low-mineralized areas in the mature-mineralized bone matrices (Fig. 4A). The osteocyte density (N.Ot) in loose periprosthetic samples was 1334.6 ± 111.4 cell/mm2, which was significantly higher than those of unloose periprosthetic samples (707.6 ± 61.1 cell/mm2; p < 0.001). The deposition of tetracycline was seen mainly as double or single linear lines. The linear lines appeared as clear but occasionally as vague double- or single-labeled lines. In addition, patchy and/or diffuse labelings also were observed ( Figs. 5A–5C). Active linear tetracycline deposition and osteoid/low-mineralized bone matrix formation were observed frequently in the periprosthetic bone, which were adjacent to monocyte/macrophage cell sheets in the granulomatous tissues ( Figs. 1A, 6A, and 6B). Tetracycline-labeled woven bone matrices seemed sometimes to be eroded soon after formation (Fig. 7).

Figure FIG. 1.

(A) Periprosthetic bone sample adjacent to loose and unloose interface tissue sample by confocal laser scanning microscopy. In the bony samples adjacent to loose interface, granulomatous connective tissues (Gr), including monocyte/macrophages, fibroblasts, and vascular endothelial cells, were observed adjacent to the retrieved bones, and infiltration of granulomatous cell components into the periprosthetic bone also was found (*). Scattered low-mineralized areas, which were stained as orange, also were found in the green-stained mature-mineralized bone matrices (arrows). Increase of tetracycline-labeled bone, osteoid (red) and low-mineralized (orange) areas, and osteoblast lining were found adjacent to granuloma cell components. Linear tetracycline deposition was observed as a yellow or green-yellow line (arrowheads). (B) Unloose interface; tetracycline-labeled bone surface, osteoid/low-mineralized area, and osteoblast lining also were observed in the bony tissues adjacent to unloose interface; however, they were low when compared with the periprosthetic bones adjacent to loose interface. White scale bars represent 50 μm.

Figure FIG. 2.

Osteoclasts (arrows) were observed in the resorption lacunae formed in the mature-mineralized periprosthetic bone samples from loose hip joint. A white scale bar represents 10 μm.

Figure FIG. 3.

Differential interference constant image transmitted light in the system was applied to evaluate whether the bone that is formed next to the implant was woven or lamellar. (A) The presence of lamellar structure was observed in the periprosthetic bone adjacent to interface tissue, but it was irregular, mosaic, and sometimes intervened by and/or coupled with woven bones, which was identified as the area of no lamellar structure. (B) Lamellar structures were observed irregularly but not so complicated in the periprosthetic bone adjacent to thin fibrous interface tissue when compared with loose periprosthetic samples.

Figure FIG. 4.

(A) Osteocytes, which had abundant dendritic cytoplasmic projections of bone canaliculi, were observed in the scattered low-mineralized area in the mature-mineralized bone matrices in the sample from loose hip prosthesis. (B) Osteocytes in unloose and well-fixed interface. Bone canaliculi were not abundant when compared with osteocytes in loose hip prosthesis. To observe cell morphology in the bone matrix, the confocal system displayed osteocytes and bone canaliculi as a red color with black background. White scale bars represent 10 μm.

Figure FIG. 5.

Tetracycline labeling (yellow or green-yellow) patterns in loose interface samples. (A) Clean (*) or vague (**) double labeling, (B) patchy labeling (***), and (C) diffuse labeling, appeared in mosaic staining by green and yellow.

Figure FIG. 6.

(A) Osteoid/low-mineralized bone matrix in the loose interface samples. Wide osteoid/low-mineralizedarea containing double tetracycline labeling (arrows) and osteoblast lining (arrowheads), which were adjacent to monocyte/macrophage sheets. (B) Osteoblast lining and osteoid. To observe the osteoblasts (arrowheads) clearly, the confocal laser scanning system displayed osteoid as yellow and cells were green.

Figure FIG. 7.

Eroded bone surface (arrowheads) containing double tetracycline labeling (arrows). This indicated that bone matrix was resolved soon after formation. A white scale bar represents 10 μm.

Periprosthetic bones adjacent to unloose interface:

In samples from well-fixed implant-to-bone interface, a thin layer of fibrous tissue usually was observed adjacent to the bony tissues (Fig. 1B), but occasionally small amounts of monocytes/macrophages and fibrovascular tissue components were observed. Although tetracycline-labeled bone surface, osteoid/low-mineralized area, and osteoblast lining also were observed in the bony tissues adjacent to unloose interface, they were low when compared with the periprosthetic bone obtained from loose interface. Lamellar structures were irregular but not so complicated in the periprosthetic bone of unloose interface when compared with loose periprosthetic bone (Fig. 3B). Osteocytes with abundant dendritic cytoplasmic projections in bone canaliculi, of the low-mineralized area, were less frequent in the well-fixed periprosthetic bones (Fig. 4B). A linear type of tetracycline deposition was the dominant pattern.

Bone histomorphometric analysis

In structural histomorphometric analysis bone volume (BV/TV) was 75.1 ± 5.7% and 72.8 ± 9.8%, and void volume (Vd.V/TV) was 24.9 ± 5.7% and 27.2 ± 9.8% in loose and unloose interfaces, respectively. They were not statistically different (BV/TV, p = 0.478; Vd.V/TV, p = 0.478). Mature-mineralized bone volume (M-Md.V/BV) was 54.6 ± 12.9% in loose interface, which was less than in unloose interface (80.6 ± 10.0%; p < 0.001). Low-mineralized bone volume (L-Md.V/BV) was 39.3 ± 12.2% and 17.6 ± 9.1%, and osteoid volume (OV/BV) was 6.1 ± 2.2% and 1.8 ± 0.9% in loose and unloose interfaces, respectively, both of which were significantly increased in loose periprosthetic bone samples (L-Md.V/BV, p < 0.001; OV/BV, p < 0.001). Osteoid thickness (O.Th) was increased more significantly in loose interface (27.4 ± 1.5 μm) than in unloose interface (12.6 ± 0.7 μm; p < 0.001; Table 2I).

Table Table 2.. Histomorphometric Analysis of Periprosthetic Bone Samples
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In surface histomorphometric analysis, osteoid surface (OS/BS), osteoblast surface (Ob.S/BS), eroded surface (ES/BS), and osteoclast surface (Oc.S/BS) were all increased in loose interface when compared with unloose interface (OS/BS: 25.8 ± 8.7% vs. 16.8 ± 3.6% and p = 0.017; Ob.S/BS: 12.0 ± 4.6% vs. 8.4 ± 2.3% and p = 0.064; ES/BS (%): 14.7 ± 5.1 vs. 9.8 ± 2.4% and p = 0.027; Oc.S/BS: 2.6 ± 0.9% vs. 1.2 ± 0.7% and p = 0.003; Table 2II).

Basic dynamic histomorphometric analysis disclosed that MAR was higher in loose interface (1.7 ± 0.7 μm/day) than in unloose interface (1.1 ± 0.3 μm/day; p = 0.044). Mineralizing surface (MS/BS) also was increased in loose interface (25.1 ± 9.2%) when compared with unloose interface (13.0 ± 7.1%; p = 0.025; Table 2III). In derived dynamic histomorphometric analysis, mineralization lag time (Mlt) was 17.6 ± 1.6 day in loose interface, which was not statistically different from that in unloose interface (15.8 ± 0.7 day; p = 0.328). Surface referent BFR (BFR/BS) was higher in loose interface (0.1783 ± 0.029 mm2/mm3 per day) than in unloose interface (0.0565 ± 0.006 mm2/mm3 per day; p = 0.002). Bone volume referent BFR (BFR/BV) also was increased more in loose interface (216.3 ± 35.6%/year) than in unloose interface (69.4 ± 7.1%/year; p = 0.002; Table 2IV).

DISCUSSION

Foreign body reaction in the granulomatous interface tissue located between bone and implant is known as an important cause of periprosthetic weakening and osteolysis. Foreign body-type adverse host reaction, together with mechanical factors, such as initial stability, type of materials, and load transfer to periprosthetic bone,(11) leads to loosening. Monocytes/macrophages, fibroblasts, and vascular endothelial cells were the main cell types in synovial-like interface membrane, where overproduction of various chemical mediators, cytokines, and proteinases have been considered to cause recruitment of osteoclast precursors and bone resorption around loose implants.

However, despite intensive research, the precise involvement of osteoclasts and osteoblasts, supposed to be recruited as a result of local production of cytokines and prostanoids, has not been clarified completely yet. Reports on periprosthetic bone analysis are few.(31–35) In particular, little is known regarding precise periprosthetic bone remodeling and bone quality. However, this seems to be one of the important key factors, when considering the biological mechanisms responsible for osteolysis and implant loosening, because periprosthetic bone contributes to the support of implant, and better osseointegration between bone and implant is essential for longer survivorship of the total hip joints.

This study revealed not only increased osteoclastic bone resorption in the periprosthetic bone adjacent to loose interface tissues, but also increased immature bone matrices (osteoid/low-mineralized bone matrix area), elevated MAR and BFR with active osteoblast lining, and abundant bone canaliculi projection of osteocytes in low-mineralized area in situ. This also is confirmed by the presence of irregular and mosaic lamellar structure, sometimes intervened by and/or coupled with woven bone by differential interference constant image transmitted light. They clearly relate to periprosthetic bone remodeling adjacent to granulomatous interface, as was shown by a comparison to control bony tissues adjacent to well-fixed interface. The data obtained by microscopic observation and bone histomorphometry suggest that active osteoclastic bone resorption and immature mineralization are coupled with monocyte/macrophage-mediated foreign body-type granuloma. Mineralization lag time was not significantly different in the study groups, but they were both higher when compared with those in the analyses of the normal population.(28) This result, combined with increased BFR, suggests that periprosthetic bone remodeling around artificial hip joint implants is highly activated. In addition, once foreign types of unfavorable host reaction occur between bone and implants, its bone remodeling process is forced to be more accelerated and results in formation of fragile bone. Thus, bone quality becomes immature and poor. This phenotype of periprosthetic bone in loose hip joints is considered to contribute to loss of implant support and osseointegration between bone and implants, thus loosening and osteolysis may proceed around total hip joints.

It is also of interest that active linear tetracycline deposition and immature bone (osteoid/low-mineralized bone matrix) formation were frequently observed adjacent to monocyte/macrophage cell sheets in the granulomatous tissues. It may suggest bipolar potential of monocytes/macrophages; they not only produce osteoclastic cytokines such as interleukin-1 (IL-1), IL-6, tumor necrosis factor α (TNF-α), and macrophage colony-stimulating factor (M-CSF) contributing to osteoclastogenesis and activation of osteoclast, but also stimulate osteoblast and woven bone matrix production, via IL-1, epidermal growth factor (EGF), transforming growth factor β (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), and insulin-like growth factors (IGFs(17) and increase survival rate of osteoblast via TGF-β and IL-6.(36) However, such bone matrices are not mineralized enough and of high enough quality to support implant. In addition, abundant bone canaliculi/projections of osteocytes in low-mineralized area in situ may implicate cell-cell interactions and osteocytic mineral regulation under stimulation of biological mediators combined with mechanical cyclic loading; such findings indicate a role for osteocytes in situ in various pathological bone states and in bone turnover.(36–41) Various types of tetracycline deposition patterns observed in periprosthetic bone around loosened implants support the hypothesis that biological mediators combined with mechanical cyclic loading may contribute to disturbances in the local bone turnover. Patchy and/or diffuse tetracycline depositions also may represent the repair phase of microfracture trauma, because of fragile bone quality and overmechanical stress from implant to bone when bearing weight.

In this study, the confocal laser microscopy system combined with differential interference constant image transmitted light was applied. It has higher resolution with computer-regulated clear morphological view superior to that of conventional microscopy with capability of three-dimensional analysis. This seems to enable more accurate histomorphometric analysis even with thicker mineralized sections without any damage of structure, for example, by decalcification and overgrinding in sample processing.(24,25)

In conclusion, high-turnover periprosthetic bone remodeling and immature bone formation are seen around loose cemented total hip prostheses with a confocal laser microscopy system, which seems to be a result of cellular host response combined with inappropriate cyclic mechanical loading. This type of bone remodeling is unique; it is not seen in normal conditions and may not be seen in any other pathological conditions. Thus, periprosthetic bone remodeling in loose hips is dependent on the host response relating to artificial materials, and the fragile bone quality may contribute to the periprosthetic connective tissue weakening and osteolysis in the process of hip prosthesis loosening. Further investigation of biomaterial research in the host-implant junction may, in the future, provide a more suitable condition for materials and contribute to the advancement in the treatment and restoration of musculoskeletal system.

Acknowledgements

We thank Ms. Eiko Saito, B.A., Yamagata University, for excellent technical assistance in staining and making tissue sections, and Ms. Hiroko Katoh, B.A., Division of Application Engineer System, Leica, Tokyo, for excellent technical assistance in the observation of confocal laser scanning microscopy. This work was supported in part by the Grant-in-Aid for Scientific Research (B), 1996–1998; Grand-in-Aid for International Scientific Research (Yamagata University-University of Helsinki Joint Research), 1999-2000; the Ministry of Education, Science, Sports and Culture; Grant-in-Aid for Musculoskeletal Research, Yamagata Health-Support Foundation, Japan, 1999-2000; the HYKS TYH 0056 evo grant, and the Finnish Academy, Finland.

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