Bone, Vol. 16, No. 1
lanuary 1995:%15
ELSEVIER
Stimulation of Osteoinduction in Bone Wound Healing by
High-Molecular Hyaluronic Acid
T. SASAKI’
and C. WATANABE’
’ Departmenf of Oral Anatomy, School of Dentistry, Showa lJniversi@, Tokyo, Japan
’ Department df Dentistry and Oral Surgery, N&ma1 Mito Hospital,. Mito I JLlp&
roles in the wound healing of various tissues, including bone
(Mustoe et al. 1987; Bab et al. 1988; Brown et al. 1988). These
growth factors appear to stimulate the early phases of wound
healing, cell differentiation, and increased primary matrix production, rather than remodeling and maturation processes (Mustoe et al. 1987; Brown et al. 1988).
One of the major glycosaminoglycans,
hyaluronic acid (HA),
recently has been reported to increase osteoblastic bone formation in vitro through increased mesenchymal cell differentiation
and migration (Pilloni & Bernard, 1992). Locally applied highmolecular HA also has been shown to stimulate differentiation
and migration of mesenchymal and muscular cells in vivo (Toole
& Trelsted 1971; Feinberg & Beebe 1983; Nettelbladt et al.
1989; Bray et al. 1991; Krenn et al. 1991). In general, a high
concentration of HA has been demonstrated in tissue repair, and
the largest amounts of HA were found in the extracellular matrix
of musculoskeletal and dermal tissues (Fraser & Laurent 1989).
The therapeutic use of highly purified HA in human and veterinary medicine now is being advocated for applications such as
idiopathic or experimentally
induced osteoarthritis,
viscosurgery, and viscosupplementation
(Balazs & Denlinger 1989;
Kikuchi et al. 1993). Therefore, a recently developed formula
for autologously prepared HA is expected to provide a potential
means of accelerating new bone formation in healing of bone
wounds. Using long bones, many investigators have examined
morphologically the healing processes that follow mechanical
removal of bone marrow and have clarified the time course of
sequential healing processes in bone wounds (Branemark et al.
1964; Amsel et al. 1969; Watanabe et al. 1992; Furusawa 1993).
The present study is the first designed to evaluate morphologically the effects of elastoviscous,
highly purified,
highmolecular HA on bone wound healing in rat femurs.
To study the osteoinductive action of hyaluronic acid (HA),
we examined the effects of applying an elastoviscous highmolecular HA preparation on bone wound healing after bone
marrow ablation. The middiaphyses of cortical bones from
rat femurs were perforated with a round bar, and excavated
marrow cavities were filled immediately with high-molecular
HA. Bone marrow ablation without HA was used to prepare
controls. On post-ablation days 1, 2, 4, 7, and 14, animals
were perfusion-fixed
with an aldehyde mixture, and dissected femurs were examined by means of light, transmission-, and scanning-electron
microscopy. In controls, the
wounded marrow cavities were first filled with blood and
fibrin clots (days 1 and Z), then with granulated tissues containing macrophages, neutrophils, and ilbroblastic cells (day
4). New bone formation by differentiated osteoblasts was observed at 1 week post-ablation; at 2 weeks, the perforated
cortical bones and marrow cavities were filled mostly with
newly formed trabecular bone. In bones to which HA had
been applied, new bone formation already had been induced
by day 4 on both the peri- and endosteal surfaces of the
existing cortical bones. At 1 week post-ablation, marrow cavities were completely filled with newly formed trabecular
bones, in which active bone remodeling by osteoblasts and
osteoclasts had occurred. Granulated tissues were replaced
rapidly by normal marrow cells. These results suggest that
high-molecular HA is capable of accelerating new bone formation through mesenchymal cell differentiation in bone
wounds. (Bone 16:9-15; 1995)
Key Words: Hyaluronic
Wound healing.
acid; Osteoinduction;
Bone marrow;
Introduction
Materials and Methods
Normal healing of bone is characterized by the integrated actions
of different cells, and its processes are divided into broad sequential phases of inflammation, proliferation, and migration of
osteogenic cells, and production and remodeling of trabecular
bones (Branemark et al. 1964; Amsel et al. 1969; Patt & Maloney 1975; Watanabe et al. 1992; Furusawa 1993). Recent in
vivo and in vitro studies have suggested that various autocrine
and/or paracrine growth factors present in serum play important
Female Sprague-Dawley strain rats, 10 weeks old, were used for
this experiment. Under general anesthesia with ethyl ether, either
the right or the left femur was exposed surgically, round perforations were formed in the mid-diaphyses of cortical bones, and
adjacent bone marrow was excavated with a sterile, invertedcone round bar (1 mm in diameter). After flushing with sterile
physiological saline, the hemorrhage was controlled with sterile
cotton pellets, and the bone cavities were filled immediately with
an elastoviscous
solution of high-molecular
HA (NRD 101,
kindly provided by Japan Roussel Co. Ltd., Tokyo). NRD 101 is
a streptococcal product containing sodium hyaluronate (1900
KDa molecular weight). Its pH is 6.8-7.8, and it contains less
than 0.04% protein. Its viscosity is 500 cps (1500 KDa MW) to
Address for correspondence and reprints: Dr. T. Sasaki, Department of
Oral Anatomy, School of Dentistry, Showa University, I-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142, Japan.
0 1995 by Elsevier Science Inc.
9
&X756-3282/95/$9.50
8756-3282(94)00001-G
10
T. Sasaki and C. Watanabe
Stimulation of osteoinduction
by hyaluronic
Bone, Vol. 16, No. 1
January 1995:+15
acid
1000 cps (2000 KDa MW). As an experimental control, the
perforated and excavated marrow cavities in other rats were
filled with only blood clots. After the perforating procedure, the
periosteum, muscles, and skin were sutured.
On days 1, 2, 7, and 14 after surgical ablation of the bone
marrow, animals (three rats per each control or experimental
group) were perfusion-fixed
through the left ventricle with a
mixture of 1% glutaraldehyde and 1% formaldehyde in a 0.1 M
sodium cacodylate buffer (pH 7.3). Dissected femurs were then
demineralized in 10% EDTA-2Na (pH 7.3) for about 1 month at
4”C, cross-cut into segments including perforated areas, washed
overnight in 0.1 M sodium cacodylate buffer, and post-fixed
with 1.5% potassium ferrocyanide-reduced
1% osmium tetroxide
for 3 h at 4°C. They were further block-stained with ethanolated
1% uranyl acetate, dehydrated through a graded ethanol series,
and embedded in Quetol 812 (Nisshin EM Co. Ltd., Tokyo,
Japan). Ultrathin sections were cut with a diamond knife on a
Reichert-Jung Ultracut OmU-4; stained with tannic acid, uranyl
acetate, and lead citrate; and examined with an Hitachi H-800
electron microscope at 75 kV. Sections 1 pm thick were prepared with the same microtome, stained with toluidine blue, and
examined with an Olympus BHS light microscope (LM).
For scanning-electron
microscopic (SEM) observation, fixed
femurs were cut longitudinally and placed in 5% sodium hypochlorite solution for about 2 h at room temperature to dissolve the
soft tissues attached to cortical bones. The cortical bones were
conductive-stained with 1% osmium tetroxide solution at 4°C for
4 h, dehydrated through a graded ethanol series, and criticalpoint dried using liquid CO,. The tissue samples were then ionsputter-coated with platinum and examined, from the bone marrow site, with an Hitachi S-430 SEM at 15 kV.
Results
Control Bone
The regenerative processes that occur in bone marrow after ablation have been described precisely in previous morphological
studies (Branemark et al. 1964; Amsel et al. 1969; Watanabe et
al. 1992; Furusawa 1993). Briefly, by l-2 days after ablation,
the wounded bone marrow had become filled with a necrotic
tissue consisting of blood and fibrin clots (data not shown). By
day 4, numerous neutrophils, macrophages, mesenchymal cells,
spindle-shape fibroblastic cells, and blood vessels could be seen
in the wounded bone marrow (Figure la). On both LM and SEM
observations, no new bone formation was observable in either
the marrow cavities or existing cortical bone surfaces (Figures
la, lb) at the 4-day point. At 1 week after ablation, trabecular
bone formation was clearly visible in marrow cavities (Figure
2a). This trabecular bone formation seemed to extend contiguously from the existing cortical bone surface toward the perforated areas and marrow cavities (Figures 2a, 2b). At 2 weeks,
the marrow cavities were completely filled with newly formed
bone trabeculae and normal marrow cells (Figures 3a, 3b). Osteoclastic resorption of newly formed trabecular bone during
bone remodeling also was observed in this phase (data not
shown).
Bone Response to HA Application
As in control bones, by l-2 days after ablation, the excavated
bone marrow had become filled with blood and fibrin clots (data
not shown). By day 4, numerous spindle-shape fibroblastic cells
and polygonal or cuboidal cells, as well as neutrophils and macrophages, were distributed densely in the wounded bone marrow
(Figure 4a inset). At the ultrastructural level, spindle-shape fibroblastic cells observed throughout the bone marrow were surrounded by dense bundles of collagen fibers (Figure 4a). As in
osteoblastic cells described below, these fibroblastic cells had a
well-developed cytoplasmic organization for protein synthesis,
consisting of Golgi apparatus and cisterns of the rough-surface
endoplastic reticulum (RER) (Figure 4a). However, they produced no calcifying structures. These findings in the wounded,
HA-applied bone marrow were almost identical to those observed in control bones at 4 days (Figures la and 4a inset).
However, on the periosteal surfaces, cuboidal or polygonal
osteoblastic cells appeared to form a cell-rich, well-vascularized
reorganizing tissue, the periosteum (Figure 4b inset). The periosteum increased in thickness during the course of wound healing. Osteoblastic cells were arranged relatively irregularly, but
partially formed a regularly arranged single cell layer (Figure 4b
inset). Bone matrix-like substances also were deposited over the
existing cortical bone surfaces (Figure 4b inset). At the ultrastructural level, cuboidal or polygonal osteoblastic cells apparently produced bone matrix, consisting mainly of type I collagen, over the existing cortical bone surfaces (Figure 4b). Osteoblastic cells possessed numerous RER cisterns throughout the
cytoplasm and a well-developed Golgi apparatus in the perinuclear cytoplasm and were surrounded by thick bundles of collagen fibers (Figure 4b). Osteoblastic cells contained electrondense secretion granules, presumably containing procollagen
molecules, in the Golgi area (data not shown). These osteoblastic
cells appeared to produce calcifying globular structures 0.7-l .5
pm in diameter, which protruded into the matrix adjacent to
existing cortical bones (Figures 4b, 4c). These calcifying globular structures consisting of electron-dense granular material in
demineralized sections were connected with surrounding collagen fibers (Figure 4~). These globules further fused with each
other, thus producing new bone matrix over the existing cortical
bone surfaces (Figures 4b, 4~). Osteoblastic cells extended long
cytoplasmic processes toward newly formed bone matrix via a
thin osteoid layer (Figure 4b). Examination with SEM also
clearly revealed that a thin layer of newly formed bone was laid
down over a portion of the existing cortical bone surfaces at both
the peri- and endosteal sites (Figure 4d). Shallow osteocytic
lacunae were clearly visible in this newly deposited bone layer
(Figure 4d inset). However, these newly formed bone layers
still did not extend toward the marrow cavity to produce trabecular bone.
By 1 week after ablation, even at the LM level, increases in
newly formed cortical and trabecular bone were identified easily
along the existing cortical bone near the perforated areas (Figures 5a, 5b). Osteoclastic resorption also was observed along
the newly formed trabecular bone (Figure 5b inset). The marrow cavities were already filled with normal marrow cells (Figure 5). On SEM observation, newly formed bone trabeculae
extended throughout the excavated marrow cavities (data not
shown). By 2 weeks after ablation, a marked increase in cortical
bone formation from both the peri- and endosteal bone surfaces
was evident (Figure 6a). In newly formed trabecular bones in
the marrow cavity, shallow osteocytic lacunae and resorption
lacunae were distributed randomly throughout the bone surfaces,
suggesting the occurrence of active bone remodeling (Figures
6b, 6~).
Discussion
The present study was designed to evaluate whether highmolecular HA has the effect of generating wound strength in
bone. Normal wound healing occurs in bone through three se-
Bone, Vol. 16, No. 1
January 1995:9-15
__lll___-
Stimulation
T. Sasaki and C. Watanabe
of osteoinduction by byaluronic acid
11
-
Control. dav 4
ntrol. 1 wee
Figure 1. Light micrograph of control bone marrow at 4 days after ablation (a). SEM image of cortical bone surface around perforation observed from
the bone marrow site (b). There is no indication of new bone formation in bone surface around the perforated area (b). Magnification: a, x 150; b, ~35.
Figure 2. Control bone marrow at 1 week after ablation. New bone formation (arrows) in bone marrow from the existing cortical bone surface is clearly
visible in both LM (a) and SEM micrographs observed from the bone marrow site (b). Magnification: a, x75; b, x 35.
Figure 3. At 2 weeks after ablation, the control bone marrow is completely filled with newly formed trabecular bone and normal marrow cells in both
LM (a) and SEM micrographs observed from the bone marrow site (b). Magnification: a, x75; b, x35.
12
T. Sasaki and C. Watanabe
Stimulation of osteoinduction
by hyaluronic
acid
Bone, Vol. 16, No.
January 19959-15
1
Figure 4. HA-treated bone marrow at 4 days after ablation. Numerous spindle-shape cells and cuboidal cells have accumulated in the wounded marrow
cavity (a, inset). Electron micrograph shows fibroblastic cells (FC) and collagenous matrix in the marrow cavity (a). On the periosteal surface,
osteoblastic cells form a new bone matrix (arrows) over the existing cortical bone surface (CB) (b, inset). Arrowheads indicate blood capillaries in the
periosteum (b, inset). Ultrastructural view shows an osteoblastic cell (OC) producing collagenous bone matrix (arrows) over the existing cortical bone
(CB) (b). (c) shows a higher magnification of calcifying globular structures (asterisks) and associated collagen fibers. (d) SEM image of new bone
formation (arrows) over the cortical bone surface at the endosteal side around the perforation. Higher SEM image of newly formed bone matrix shows
shallow, newly formed osteocytic lacunae (arrowheads) (d, inset). CP: cell processes. Magnification: a, X7500; a inset, X300; b, X7500; b inset,
x600; c, ~25,000; d, X40; d inset, X 130.
Bone, Vol. 16, No. 1
January 1995:9-15
T. Sasaki and C. Watanabe
Stimulation of osteoinduction by hyaluronic acid
13
Figure 4. Continued
quential phases: postoperative inflammation, proliferation and
migration of mesenchymal cells with production of bone matrix,
and bone remodeling (Branemark et al. 1964; Amsel et al. 1969;
Patt & Maloney 1975; Watanabe et al. 1992; Furusawa 1993).
Such early sequential reorganization in wounded bone marrow is
basically identical to that seen in the presently examined bone, to
which HA had been applied. However, new bone formation in
wounded, HA-applied bone marrow was apparently earlier than
that in untreated controls. Although new bone formation was
observed at 6 or 7 days after bone marrow ablation in previous
similar experiments,
application
of high-molecular
HA to
wounded bone marrow accelerated new bone formation in the
peri- and endosteal surfaces by post-ablation day 4. Bone marrow is a highly vascularized hematopoietic tissue and contains
branching vascular sinuses lying in fibroblastic stromal cells
(Weiss 1976). In this regard, stimulation of angiogenesis by
partially degraded HA was reported to be due to the direct action
of HA on endothelial cells, possibly via a receptor-mediated
mechanism (West & Kumar 1989). If HA application did, in
fact, increase blood-vessel invasion into the wounded bone marrow, it might have affected new bone formation indirectly during
the process of bone wound healing.
In bone marrow, the osteogenic precursor cells are regarded
as belonging to the fibroblast colony-forming family of cells,
which have osteogenic properties (Ashton et al. 1984). In HAapplied bones, accumulations of differentiating fibroblastic and
bone matrix-producing osteoblastic cells were observed at day 4
in both the periosteum and endosteum, as well as in marrow
cavities. These cells may be induced through mesenchymal cell
differentiation. It is of interest that cell-associated HA-binding
proteins were isolated from locomoting fibroblasts (Turley
1989). This HA-binding protein may represent a binding site that
transduces HA’s effects to locomoting fibroblasts. In fact, Samuel et al. (1993) recently reported that TGF+stimulated
fibrosarcoma cell locomotion involved the HA receptor and HA.
Locally applied high-molecular HA has been shown to have
a positive effect in vivo on differentiation, migration, and invasion of various cell types such as cornea1 mesenchymal cells
(Toole & Trelstad 1971), blood-vessel cells (Feinberg & Beebe
1983; West & Kumar 1989), wing bud myoblasts (Krenn et al.
1991), and fibroblasts and macrophages in wound repair responses (Nettelbladt et al. 1989; Bray et al. 1991). Toole &
Trelstad (197 1) also suggested that HA might provide a suitable
substratum for the migration of influenced mesenchymal cells,
often an important early phase of morphogenesis.
The enormously extended molecular configuration of HA and its highly
negative charge also are presumed to be important factors (Toole
& Trelstad 1971). In fact, high-molecular
HA (MW: lOOO10,000 KDa) exhibited a strong positive effect on myoblast migration within the avian embryonic wing bud (Krenn et al.
1991). Because a similar effect was reproducible with dextran
sulfate, which had physicochemical properties similar to those of
HA, HA’s influence on myoblast migration was attributed to its
physicochemical properties.
In HA-applied bones, new bone formation was detected first
as the production of clusters of globular structures, consisting
primarily of granular substances containing few collagen fibers.
It was established previously in both undemineralized and demineralized sections that these calcifying globular clusters consist of apatite crystals and granular organic substances (Yanagisawa et al. 1988). These.newly formed globular calcified repair tissues, located over existing cortical bone, are therefore
thought to be induced in the initial phase of woven bone formation over the existing cortical bone lamellae. Thus, highmolecular HA seems to have a positive effect on the generation
of new bone after bone wounds. However, it is uncertain whether HA has a direct or indirect effect(s) on osteogenic cells. Recently, low-molecular HA (MW: 30 KDa) was reported to increase bone formation in vitro through increased mesenchymal
cell migration and differentiation (Pilloni & Bernard 1992). Al-
14
T. Sasaki and C. Watanabe
Stimulation of osteoinduction
by hyaluronic
acid
Bone, Vol. 16, No. 1
January 19959-15
Figure 5. HA-treated bone marrow at 1 week after ablation. New bone formation (arrows) is shown in the periosteum (a) and in the marrow cavity
(b). Figure Sb inset shows osteoclasts (arrowheads) in bone trabeculae. Newly formed bone trabeculae and marrow cells are evident in the marrow
cavity (c). Magnification: a, X 150; b, X75; b inset, X300; c, X600.
Figure 6. HA-treated bone marrow at 2 weeks after ablation. Increase in the formation of newly formed cortical bone layers over the existing cortical
bone (CB) at the periosteal (single arrow) and endosteal sites (double arrows) (a). Arrowheads indicate perforation line (a). Newly formed trabecular
bone shows both osteoblasts and osteoclasts (arrowheads) (b). SEM images of newly formed trabecular bone, in which shallow, newly formed
osteocytic lacunae and resorption lacunae (arrows) are visible (c). SEM observation was done from the bone marrow site. Magnification: a, ~75; b,
x 150; c, x 125.
Bone, Vol. 16, No. 1
January 19959-15
though high-molecular HA showed less influence on bone formation in vitro (Pilloni & Bernard 1993, this may be due to the
high viscosity of high-molecular HA in vitro. Because applied
HA is thought to be degraded into poly- and monosaccharide
components in situ, high-molecular HA may have the beneficial
effect of retaining itself within the local tissue to which it was
applied.
High-molecular HA may have another beneficial effect on
new bone formation in bone wound healing. It is well known that
various locally released growth factors have stimulatory effects
on tissue repair. For instance, epidermal growth factor and transforming growth factor-beta (TGF-l3) strongly stimulate collagen
production and fibroblast proliferation in vivo (Mustoe et al.
1987; Brown et al. 1988). In particular, TGF-l3 strongly increases bone formation in vivo and in vitro by stimulating the
proliferation and differentiation of osteogenic cells (Noda &
Camilliere 1989; Hock et al. 1990; Mackie & Trechsel 1990;
Marcelli et al. 1990). Bab et al. (1988) also reported that the
partially purified factor obtained from regenerating bone marrow
stimulated fetal long-bone elongation and DNA synthesis in osteoblastic calvarial cells in vitro. HA may have a role in maintaining these factors within the local environment. Taken together, these data suggest that high-molecular HA functions by
effectively retaining osteoinductive growth factors within the local environment by virtue of its physicochemical properties;
it is capable of accelerating new bone formation during
bone wound healing through stimulation of osteogenic cell
differentiation.
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Date Received:
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Date Revised: May 25, 1994
Date Accepted: June 27, 1994