X.-Juan Sun Maxillary sinus ﬂoor elevation using a...

Maxillary sinus floor elevation using atissue-engineered bone complex withOsteoBonet and bMSCs in rabbits

X.-Juan SunZ.-Yuan ZhangS.-Yi WangS. A. GittensX.-Quan JiangL. Lee Chou

Authors’ affiliations:X.-Juan Sun, Z.-Yuan Zhang, S.-Yi Wang,Department of Oral and Maxillofacial Surgery,School of Stomatology, Ninth People’s Hospital,School of Medicine, Shanghai Jiao Tong University,Shanghai, ChinaS. A. Gittens, Faculty of Pharmacy andPharmaceutical Sciences, University of Alberta,Edmonton, Alta, CanadaL. Lee Chou, School of Dental Medicine, BostonUniversity, MA, USA

Correspondence to:X.-Quan JiangOral Bioengineering LabSchool of StomatologyNinth People’s HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai 200011ChinaTel.: þ 86 21 63135412Fax: þ 86 21 63135412e-mail: [email protected]

Key words: bone marrow stromal cells (bMSCs), inorganic material (OsteoBonet), rabbits,

sinus floor elevation, tissue engineering

Abstract

Objectives: To evaluate the effects of maxillary sinus floor elevation by a tissue-engineered

bone complex with OsteoBonet and bone marrow stromal cells (bMSCs) in rabbits.

Material and methods: Autologous bMSCs from adult New Zealand rabbits were

cultured and combined with OsteoBonet at a concentration of 20 � 106 cells/ml

in vitro. Twenty-four animals were used and randomly allocated into groups. For each

time point, 16 maxillary sinus floor elevation surgeries were made bilaterally in

eight animals and randomly repaired by bMSCs/material (i.e. OsteoBonet), material,

autogenous bone and blood clot (n¼4 per group). A polychrome sequential

fluorescent labeling was also performed post-operatively. The animals were sacrificed

2, 4 and 8 weeks after the procedure and evaluated histologically as well as

histomorphometrically.

Results: New bone area significantly decreased from weeks 2 to 8 in the blood clot group,

while bone area in the autologous bone reduced from weeks 4 to 8. In both groups, a

significant amount of fatty tissue appeared at week 8. Accordingly, augmented height in

both groups was also significantly decreased from weeks 2 to 8. The bone area in the

material-alone group as well as in the bMSCs/material group, on the other hand, increased

over time. Significantly more newly formed bone area and mineralization was observed in

the center of the raised space in the bMSCs/material group than in the material-alone

group. The augmented height was maintained in these two groups throughout the course

of this study.

Conclusion: These results suggest that OsteoBonet can successfully be used as a bone graft

substitute and that the combination of this material with bMSCs can effectively promote

new bone formation in sinus elevation.

Osseointegrated implants are considered to

be an ideal alternative to replace missing

teeth. However, the bone height from the

alveolar crest to the sinus floor at the poster-

ior maxillary region is usually not adequate

enough due to sinus pneumatization as well

as the lack of stability caused by maxillary

bone loss at edentulous sites required for

osseointegrated implantation. Among the

various techniques used to regain the height

of resorbed maxilla, maxillary sinus floor

elevation is regarded as an effective way to

restore the upper jaw (Jensen & Shulman

1996). This procedure is based on the eleva-

tion of the Schneiderian membrane from

the floor of the maxillary sinus and the

introduction of either a bone graft or a

bone substitute (Graziani et al. 2004).

Date:Accepted 28 December 2007

To cite this article:Sun X-J, Zhang Z-Y, Wang S-Y, Gittens SA, Jiang X-Q,Chou LL. Maxillary sinus floor elevation using tissue-engineered bone complex with OsteoBonet and bMSCsin rabbits.Clin. Oral Impl. Res. 19, 2008; 804–813doi: 10.1111/j.1600-0501.2008.01577.x

804 c� 2008 The Authors. Journal compilation c� 2008 Blackwell Munksgaard

Since its introduction by Tatum and

Boyne (Boyne & James 1980; Tatum

1986), the use of autogenous bone grafts

in sinus augmentation has been considered

to be the ‘gold standard’ because of their

excellent survival with loaded implants and

the degree of functionality they afford

(Kent & Block 1989; Raghoebar 1993;

Neukam 1994; Nishibor 1994). However,

harvesting autogenous bone is generally

associated with several limitations, includ-

ing morbidity, infection, pain and blood

loss. In fact, reports suggest that the com-

plication rate of autogenous iliac grafts is

8% (Ueda et al. 2001). Autografts taken

from an intraoral donor site (e.g. mandible,

tuberosities) are limited in supply (Schim-

ming & Schmelzeisen 2004; Boyne et al.

2005).

To address the foregoing limitations as-

sociated with autogenous sources of bone,

an array of alternatives, including allograft,

xenograft and synthetic materials, has been

explored. Unfortunately, these materials

are associated with their own drawbacks.

Allografts and xenografts, for example, are

susceptible to immunorejection and carry

the risk of disease transmission (Moore

et al. 2001; Simon et al. 2002; Ueda et al.

2005). Some synthetic materials, on the

other hand, have limited potential for os-

teoconduction; using them alone has

usually failed to achieve the bone volume

expected (Wiltfang et al. 2002; Engelke

et al. 2003). Highlighting the importance

as well as the difficulty associated with

finding a suitable matrix is the fact that

bone regeneration within the sinus is de-

creased due to air pressure and low vascu-

larization within the area (Smiler 1992;

Schimming & Schmelzeisen 2004).

The ideal bone substitutes should be

biocompatible, and not only be osteocon-

ductive, but osteogenic and/or osteoinduc-

tive as well. Tissue engineering, namely

the application of scientific principles to

the design, construction, modification and

growth of living tissue using biomaterials,

cells and factors, alone or in combination,

may provide a means of developing

novel synthetic materials that possess the

aforementioned characteristics (Langer &

Vacanti 1993; Sittinger et al. 1996; Chang

et al. 2004).

Bone marrow stromal cells (bMSCs)

have been regarded as multipotent cells

residing in the bone marrow. Under ade-

quate culture conditions, bMSCs can dif-

ferentiate into various lineages of

mesenchymal tissue, including bone, car-

tilage, fat, tendon, muscle and marrow

stroma (Caplan 1991; Pittenger et al.

1999). In addition, they are relatively easy

to harvest and easily expandable in vitro

(Prockop 1997). These advantages have

made bMSCs ideal seed cells for tissue

engineering. In fact, the combination of

bMSCs with three-dimensional (3D) scaf-

folds has been thought to be the most

promising strategy to facilitate bone regen-

eration (Salgado et al. 2004).

To our knowledge, there are very few

clinical or animal studies that report using

the principles of tissue engineering to aug-

ment maxillary sinus (Schimming &

Schmelzeisen 2004; Ueda et al. 2005).

The scaffolds used in these experiments

are mainly polymeric materials, while the

seed cells are mostly sourced from the

periosteum (Schmelzeisen et al. 2003;

Schimming & Schmelzeisen 2004;

Springer et al. 2006; Zizelmann et al.

2007). In light of the fact that they can be

molded into a 3D scaffold and their degra-

dation rates can be customized, the poly-

mers used in these studies, like the poly

(lactide-co-glycolide), are inherently versa-

tile. Under certain conditions, however,

the hydrolyzation of these polymers can

produce an acidic environment and cause

tissue inflammatory and foreign body reac-

tion (Bostman et al. 1990). Although syn-

thetic bone graft substitutes ought to

exhibit biomechanical properties similar

to the bone that is being used for replace-

ment (Moore et al. 2001), most organic

materials lack the mechanical competence

needed (Rose & Oreffo 2002). Some inor-

ganic materials, however, are mechanically

suitable (Moore et al. 2001).

One example of such a material is Os-

teoBonet. This novel inorganic ceramic

material has an average porosity diameter

of 100–300 mm and an interporosity dia-

meter of 350–500 mm. As its ratio of cal-

cium and phosphorus is similar to that of

normal bone tissue, this material is biode-

gradable and possesses favorable mechan-

ical properties. Moreover, the presence of

calcium, phosphorus and silicon ions in-

side the scaffold has been shown to pro-

mote osteoblastic differentiation of seeded

cells as well as cell proliferation (Sun et al.

1997; Knabe et al. 2005).

The aim of the present study was to

explore the effects of the novel inorganic

materials OsteoBonet and autologous

bMSCs in rabbit’s sinus elevation.

Material and methods

Animals

Twenty-four male New Zealand rabbits,

each weighing from 2 to 2.5 kg, were

used for this study. [All animals were

obtained from the Ninth People’s Hospital

Animal Center (Shanghai, China), and all

procedures involving the use of rabbits

were approved by the Animal Research

Committee of the Ninth People’s Hospi-

tal.] The animals were randomly allocated

into 2-, 4- and 8-week observation groups

with eight rabbits in each group. At each

time point, 16 maxillary sinus floor eleva-

tion surgeries in all eight animals were

made and randomly repaired, with the

following four groups: group A consisted

of a tissue-engineered bMSCs/OsteoBonet

(Yenssen Biotech, Jiangsu, China) complex

(four cases); group B consisted of OsteoB-

onet alone (four cases); as a positive con-

trol, group C consisted of autogenous bone

obtained from iliac bone (four cases); and as

a negative control, group D consisted of

blood clot (four cases).

bMSCs isolation, culture and osteoblasticcharacteristic tests

Three milliliters of bone marrow was aspi-

rated from the fibula of a rabbit and cul-

tured, as described previously in Jiang et al.

(2005), in Dulbecco’s modified Eagle’s med-

ium (Gibco, Grand Island, N.Y., USA) with

10% fetal bovine serum (Hyclone, PERBIO,

Auckland, New Zealand). After 5 days,

non-adherent cells were removed and fresh

medium was added. The remaining adher-

ent cells were mainly mesenchymal stromal

cells. After the first passage, the following

three supplements for inducing osteogenesis

were added: 10�8 mmol/l dexamethasone

(Dex), 50mg/ml C-ascorb and 10 mmol/l b-

glycerophosphate (Sigma, St Louis, MO,

USA). The cells were then incubated con-

tinuously at 371C in 5% CO2. The cells at

passage 2 were used in our study.

After culturing in the induced medium

for 14 days, the cells were measured by

alkaline phosphatase (ALP) staining and

the Von Kossa test, as described in Jiang

(Jiang et al. 2005). Briefly, the cells induced

Sun et al . Maxillary sinus floor elevation using a tissue-engineered bone complex

c� 2008 The Authors. Journal compilation c� 2008 Blackwell Munksgaard 805 | Clin. Oral Impl. Res. 19, 2008 / 804–813

were fixed for 10 min at 41C and incubated

with a mixture of naphthol AS–MX phos-

phate, N,N-dimethylformamide and fast

blue BB salt (ALP kit, Hongqiao, Shanghai,

China). The Von Kossa staining method

consisted of the cells being fixed in 70%

ethanol and stained with 5% silver nitrate,

5% Na2SO3 and then observed.

Preparation of cell material complex usedin vitro and in vivo

bMSCs were collected and washed with

PBS, counted and then combined with the

material (i.e. OsteoBonet) at a final con-

centration of 20 � 106 cells/ml for the si-

nus surgeries. Extra cell material

complexes were further cultured in com-

plete medium at 371C in vitro; cell attach-

ment and spreading were visually assessed

4 h as well 4 days later through scanning

electron microscopy (Philips Quanta-200,

FEI, Eindhoven, Holland).

Autogenous bone was harvested from the

iliac crest, as described by Ueda et al.

(2001). Briefly, a 15-mm incision was

made over the iliac crest after local anesthe-

sia, which exposed the ilium. A 15 � 5 mm

corticocancellous bone block was harvested

from the right iliac crest. The periosteum

and skin flap were replaced and sutured.

The corticocancellous bone block was cut

into small particles before they were grafted

to the maxillary sinus. The blood to be used

as blood clot for the experiments was col-

lected from the ear vein with a sterile

syringe and allowed to clot naturally before

the surgery.

Maxillary sinus floor augmentationprocedure

The rabbits were anesthetized with 0.5

mg/kg sodium pentobarbital intravenously.

0.5 ml of 1% lidocaine with epinephrine

(1 : 100,000) was injected subcutaneously

for local anesthesia. According to the sur-

gical method performed by Asai et al.

(2002) and Xu et al. (2003), a 2.5 cm

vertical midline incision was made and

the skin and periosteum were subsequently

raised to expose the nasal bone and nasoin-

cisal suture line. Using a round bur, two

oval nasal bone windows (8 � 4 mm) were

outlined bilaterally on the nasal bone. The

window was located approximately 20 mm

anterior to the nasofrontal suture line and

10 mm lateral to the midline. Then, fenes-

trae were made by osteotomy during con-

tinuous cooling with sterile saline solution.

Care was taken to avoid the damage to the

antral membrane, which moved back

and forth with the respiratory rhythm. A

Freer elevator (Medical equipment limited

company, Shanghai, China) was used to

gently push the membrane inward (Fig.

1a). The membrane was then raised from

the floor and lateral walls of the antrum to

provide a large compartment. One of four

different grafts, which consisted of either

bMSCs/material, material alone, autoge-

nous bone particles or blood clot, was

slightly filled without compression into

the compartment. The size of the cavity

created at this structure was standardized

by volume among the groups. Sutures were

then placed to close the periosteum and

skin (Fig. 1b and c).

Sequential fluorescent labeling

A polychrome sequential labeling method

(Roldan et al. 2004) was carried out to label

the mineralized tissue and assess the time

course of new bone formation and remodel-

ing. Two and 4 weeks after the operation,

the animals were administered with

25 mg/kg of tetracycline and 30 mg/kg of

alizarin complexon intraperitoneally, re-

spectively. Twenty milligrams per kilo-

gram of calcein green (Sigma) was

administered 3 days before the animals

were sacrificed at week 8.

General and histological observation

The rabbits were sacrificed at 2, 4 or 8

weeks after surgery, exsanguinated and

perfused via the jugular vein with 10%

buffered formaldehyde. The maxillae were

dissected and cut into smaller blocks,

which included the nasal and maxillary

sinus, then fixed in the same solution.

The block was divided in the transverse

plane at the rostrocaudal midpoints of the

osteotomy site. One half was decalcified,

embedded in paraffin, sectioned into 4-mm-

thick sections and stained with hematox-

ylin–eosin. The other half was dehydrated

gradually in ethyl alcohol and was finally

embedded in polymethymetacrylate. The

specimens were cut into 150-mm thick

sections using a microtome (Leica, Ham-

burg, Germany), and were subsequently

ground and polished to a final thickness

of about 40 mm (Donth & Breuner 1982;

Rohrer & Schubert 1992). Undecalcified

sections were observed for fluorescent la-

beling using a microscope under ultraviolet

light. Decalcified sections were subject to

histologic and histomorphometrical obser-

vations.

Histomorphometric analysis

The measurements were performed with

decalcified specimens using a personal

computer-based image analysis system

(Image-Pro Plust, Media Cybernetic, Silver

Fig. 1. Illustration of the surgical procedure: the maxillary sinus was opened and the membrane was

subsequently pushed inwards (a). Filling the grafts into the sinus (b). The area (outlined by a red line) in the

X-ray radiograph reflects the sinus (c).


806 | Clin. Oral Impl. Res. 19, 2008 / 804–813 c� 2008 The Authors. Journal compilation c� 2008 Blackwell Munksgaard

Springs, MD, USA). Four randomly se-

lected sections from the serial sections

collected from each sample were analyzed

manually. The newly formed bone area

(i.e. the percentage of newly formed bone

area in the raised area observed) and the

augmented height (i.e. the maximum

length in the augmented space) were

recorded and compared as described pre-

viously by Ueda et al.(2001) and Ohya

et al.(2005).

Statistical analysis

Statistically significant differences

(Po0.05) between the various groups and

implantation times were measured using

ANOVA and SNK post hoc. All statistical

analysis was carried out using an SAS 8.2

statistical software package (SAS, Cary,

NC, USA). All the data are expressed as

mean� standard deviation.

Results

Cell culture, ALP and Von Kossa staining

Cell clones formed 5–7 days after initial

seeding and reached confluence after ap-

proximately 12–14 days. bMSCs at passage

2 displayed the typical fibroblastic spindle-

shaped phenotype and were used for further

studies (Fig. 2a). Two weeks after culture

in osteogenic medium, areas of ALP-posi-

tive staining and calcium deposits were

observed (Fig. 2b and c), which suggested

that cells induced by Dex led to the bMSC

differentiation into osteoblastic cells: an

occurrence that is well documented in the

literature (Cornet et al. 2002, 2004).

Adhesion and spreading of bMSCs on thematerial

Under the scanning electronic microscope,

the OsteoBonet appeared porous in struc-

ture; the pores were interconnected with

each other (Fig. 3a). Four hours after the

bMSCs were combined with the material,

cells attached to the surface of the scaffold

in vitro. After 4 days, cell spreading on the

implant surfaces was observed (Fig. 3b).

Additionally, these results suggested that

the material was suitable for the proposed

in vivo studies as it facilitated bMSCs

adhesion and spreading onto its surface.

General observations

After surgery, all rabbits recovered well.

Slight post-surgical edema was observed

at the recipient site in each animal. This

disappeared 2–3 days after the procedure.

There was no sign of infection at any time.

Histological findings

At 2 weeks after implantation

In both group A (bMSCs/material) and

group B (material alone), the augmented

space was convex and newly formed trabe-

culae were mainly found close to the parent

bony wall and raised membrane (Fig. 4a

and c). In the same position of the aug-

mented area, more newly formed bone was

observed in group A (Fig. 4b) than in group

B (Fig. 4d). All trabeculae and material

particles were embedded in fibrous connec-

tive tissue.

Group C (autogenous bone) showed tra-

beculae of newly formed bone around the

grafted bone. The lacunae around the os-

teocytes were large. The newly formed

trabeculae were embedded in fibrovascular

tissue with no evidence of inflammation

(Fig. 7a and b).

In group D (blood clot), the augmented

space was concave, and newly formed tra-

beculae embedded in fibrovascular tissue

were found at the center of the cavity. No

remnants of blood clots were visible. More-

over, there was no evidence of inflamma-

tion at the site (Fig. 8a and b).


In both group A and group B, newly formed

bone found adjacent to the bony wall of the

Fig. 2. The second passage bMSCs displayed the typical spindle-shaped fibroblastic phenotype ( � 10) (a).

Alkaline phosphatase-positive stain area (b) and Von Kossa-positive stain area (c) 14 days after having been

induced by dexamethasone ( � 16).

Fig. 3. OsteoBonet: a new inorganic material with a porous structure with an average pore diameter of 200mm

(a). Four days after the bMSCs were combined with OsteoBonet, they could be seen attaching and spreading to

the inner surface of the scaffold (b).



cavity was denser than at 2 weeks (Fig. 5a

and c). More mineralized bone in the

bMSCs/material sites were also observed

in the center of the augmented area than in

the material sites alone (Fig. 5b and d).

In the autogenous bone site, the aug-

mented space filled with trabeculae that

were more mature than at 2 weeks. Appar-

ent lamellar bone structure was also ob-

served adjacent to the cortical bone wall of

the cavity.

In the blood clot site, the augmented

space appeared to be smaller than that 2

weeks after implantation. Trabeculae in

the augmented space were more mature

than that at 2 weeks.


In both group A (bMSCs/material) and B

(material alone), the augmented space re-

mained convex. Several formed bones were

observed that showed many interconnec-

tions. This newly formed bone bound to

the material particles tightly in certain

areas (Fig. 6a and c). In the bMSCs/mate-

rial sites, the thick lamellar bone was more

frequently found in the center of the space

(Fig. 6b). In comparison, the general ten-

dency of bone maturation in the bMSCs/

material sites was greater than in the ma-

terial-alone sites (Fig. 6d).

In group C (autogenous bone), cortical

bone formation was observed adjacent to

the raised membrane. The grafted bone and

newly formed trabeculae observed at 2 or 4

weeks were reduced. The cortical bone was

also embedded with fatty tissue (Fig. 7c

and d).

In group D (blood clot), the augmented

space reduced dramatically. The sinus

membrane that was raised almost turned

back, forming a sinus cavity again (Fig. 8c).

Few newly formed bones were found under

the raised sinus membrane. This area was

also embedded with fatty cells (Fig. 8d).

Fluorescence microscopy

The deposition of mineralized bone matrix

was observed in the bMSCs/material and

material-alone sites at different time points

as demonstrated by green tetracycline (yel-

low), complexon (red) and calcein green

(green) (Fig. 9a and b). All the three differ-

ent fluorescent-labeling areas in group A

(bMSCs/material) were larger than those in

group B (material alone). As evidenced by

the fluorescence of tetracycline (yellow) at

week 2 and alizarin complexon (red) at

week 4 in group A (Fig. 9a), the miner-

alization of new bone was present more

frequently in group A than in group B in

the center of the augmented area. These

data suggest that bMSCs contributed to the

enhanced mineralized area over time.

Histomorphometric analysis

Bone area

At 2 weeks, bone area of the autogenous

bone site (34.79� 7.18%) was significantly

higher than that of group A (bMSCs/

Fig. 4. Histological findings at 2 weeks after implantation. Most newly formed trabecular bone was found

along the periphery of the raised sinus and the surface of the OsteoBonet near the parent cortical bone wall in

both group A (a, b) and group B (c, d). NB, nasal bone; M, augmented sinus membrane; P, particles; B, bone.

a–d, hematoxylin–eosin; a, c, � 1.25; b, d, � 10.

Fig. 5. Histological findings at 4 weeks after implantation. There was more newly formed bone than that at 2

weeks found in both group A (a, b) and group B (c, d). NB, nasal bone; M, augmented sinus membrane;

P, particles; B, bone. a–d, hematoxylin–eosin; a, c, � 1.25; b, d, � 10.



material) (21.04� 2.67%) (Po0.05) and

group B (material) (19.22� 2.25%)

(Po0.05). No significant difference was ob-

served between other groups. At 4 weeks,

bone area of group A (23.47� 3%), group

B (22.95� 2.18%) and group C (autoge-

nous bone) (28.75� 5.8%) was all signifi-

cantly higher than that of group D (blood

clot) (15.7� 3.33%) (Po0.05). There was

no significant difference between the other

groups. At 8 weeks, the bone area of group

A (35.36� 10.57%) was significantly

higher than that of group C (19.58�2.45%) and group D (13.84� 4.03%)

(Po0.05). No significant differences were

detected between groups C and D. There

was 30% more bone area in group A than

in group B, although this was not statisti-

cally significant due to, perhaps, high in-

tra-group variation.

In the bMSCs/material site, bone area

increased significantly from 4 to 8 and 2 to

8 weeks (Po0.05). Bone area in the mate-

rial site did not show a significant increase

between any observation times. In group

C, bone area decreased over time. There

was a significant decrease in the bone area

from 2 or 4 weeks to 8 weeks (Po0.05). In

group D, from 2 weeks (27.62� 6.44%)

to 4 weeks (15.76� 3.33%) and from 2 to

8 weeks, significant bone area decrease was

also observed (Po0.05) (Fig. 10).

Augmented height

For both control groups, the augmented

height decreased significantly from 2 to 8

weeks (Po0.05). In group C (autogenous

bone group), significant differences were

observed at different times after implanta-

tion (Po0.05). In group D (blood clot), a

significant difference could be observed

between 2 and 8 and 4 and 8 weeks

after surgery (Po0.05). However, for group

A (bMSCs/material) and group B (material

alone), the augmented height was not sig-

nificantly different (Po0.05). Both groups

A (bMSCs/material) and B (material alone)

had a higher augmented height than that in

group D at each observation time point

(Po0.05); they were also significantly dif-

ferent from group C at weeks 4 and 8

(Po0.05). The augmented height in group

C was higher than in group D at 2 and 4

weeks (Po0.05), but this difference was no

longer present at 8 weeks (Fig. 11).

Discussion

The use of rabbits as a model for maxilla

sinus elevation is well-documented: rabbits

have the same ventilation with air ex-

changes through the nasal cavity as hu-

mans and a well-defined ostium opening to

their nasal cavities as well (Kumlien &

Schiratzki 1985; Scharf et al. 1995). As

air pressure causes movement of the max-

illary sinus membrane, the grafted material

in the sinus is subjected continuously to

Fig. 6. Histological findings 8 weeks after implantation. Newly formed bone with many interconnections was

observed in most of the convex augmented space in both group A (a, b) and group B (c, d).NB, nasal bone; M,

augmented sinus membrane. a–d, hematoxylin–eosin; a, c, � 1.25; b, d, � 10.

Fig. 7. Histological findings of group C (autogenous bone). Fibrovascular tissue was observed surrounding

newly formed trabecular bone 2 weeks after implantation at magnifications of � 1.25 (a) and � 10 (b).

Cortical bone, which was surrounded by fatty tissue, was observed adjacent to the raised membrane 8 weeks

after implantation at magnification of � 1.25 (c) and at � 10 (d). NB, nasal bone; M, raised sinus membrane;

B, bone.



this air pressure, which will ultimately

affect the augmented bone healing process

and structure (Garey et al. 1991; Asai et al.

2002). Under these conditions, the grafting

material is considered to be an important

determinant of the success or failure of a

bone augmentation procedure (Xu et al.

2004). Results from numerous clinical

cases have already suggested that freeze-

dried demineralized bone cannot withstand

air pressure in the sinus (Jensen & Shul-

man 1996; Block & Kent 1997; Wheeler

1997). Some studies suggest that even

alloplastic materials have problems in

these ischemic areas (Schmelzeisen &

Schimming 2003). Hence, in addition to

being capable of stimulating osteoinduc-

tion, osteoconduction and maturation of

new bone structure, while resisting resorp-

tion, maxillary sinus grafts should also be

able to provide adequate stability (Block &

Kent 1997). Blood clots and autologous

bone have been explored previously as

grafts (Block & Kent 1997; Xu et al.

2004). Consequently, these materials

were used as controls in this study.

As expected, we observed some newly

formed bone 2 weeks after blood clot

transplant. However, the new bone area

decreased significantly from 27.62% at 2

weeks to 13.84% at 8 weeks. Indeed, the

augmented height decreased significantly

from 2.92� 0.78 mm at 2 weeks to

1.43� 0.26 mm at 8 weeks (Po0.05)

and the raised sinus mucosa almost re-

turned to its original position. Despite the

abundance of growth factors known to

contribute to the new bone formation in

the blood clot graft, such as endothelial

growth factor, fibroblast growth factor, in-

sulin-like growth factor and transforming

growth factor-b (TGF-b) (Jensen et al.

1995; Smukler et al. 1995; Leghissa et al.

1999), blood clot alone was not a suitable

substitute material for sinus lift. This

finding, which is similar to that observed

in Xu et al. (2004), might be due to

the failure of the grafts to withstand

sinus pressures for an extended period of

time.

To our surprise, autogenous bone, the

gold standard, only maintained a sufficient

bone area for 4 weeks following the proce-

dure; the bone areas as well as the height of

the autograft, however, were reduced sig-

nificantly beginning from 4 to 8 weeks

(Po0.05). Here, bone was replaced by a

significant amount of fatty tissue (as ob-

served at 8 weeks), which might be the

result of the fatty marrow characteristic of

the particular animal (Ohya et al. 2005).

Fig. 9. Fluorescent micrography findings 8 weeks after implantation. As shown by the fluorescence of

tetracycline (yellow), alizarin complexon (red) and calcein green (green), the mineralization of new bone was

significantly greater in group A (a) over time than that in group B (b, � 10).

40353025201510

50

45

2 4 8

Time (weeks)

Bon

e ar

ea (

%)

Fig. 10. Implant bone area assessed at various time

points using histomorphometry. (n indicates signifi-

cant differences Po0.05).

02468

101214

2 4 8

Time (week)

Aug

men

ted

heig

ht(m

m)

bMSCs/M M Autogenous bone Blood clot

Fig. 11. Maxillary sinus augmented height assessed

at various time points using histomorphometry. (n

indicates significant differences Po0.05).

Fig. 8. Histological findings of group D (blood clot). 2 weeks after implantation, newly formed trabecular bone

was found at the periphery of the sinus (a, � 1.25). Many of these trabeculae were surrounded in fibrovascular

tissue with no evidence of inflammation (b, � 10). Eight weeks after implantation, the augmented space was

concave, the membrane raised turned back and continuous cortical bone formation was observed under the

raised sinus membrane as well as at the bony wall (c, � 1.25). Some of the newly formed trabeculae, which

were clearly lamellar, were surrounded in fatty tissue (d, � 10). NB, nasal bone; M, raised sinus membrane; B,

bone.



Similar results were reported in other stu-

dies (Watanabe et al. 1999; Wada et al.

2001). Another explanation might be that

cortical and cancellous bone in the auto-

genous bone graft could not withstand

sinus pressures for long periods of time,

and as such, start to lose their density and

height during the first several weeks (Jen-

sen & Shulman 1996). Some published

clinical data showed that the absorption

rate using autogenous bone in sinus aug-

mentation was 47% 6–7 months after

surgery (Johansson et al. 2001). Watanabe

et al. also linked the absorption rate of

different donor sources to different ratios

of cortical bone or cancellous bone (Wata-

nabe et al. 1999). For example, chin bone

grafts demonstrated greater volume main-

tenance than did the iliac bone simply

because chin bone contains more cortical

bone. Secondly, bone grafts usually need

functional loading stimulation (via im-

plants, for example) to counter the innate

response of resorbing bone implants (Schle-

gal et al. 2003; Ohya et al. 2005). In this

preliminary study, we did not provide any

functional loading stimulation. Conse-

quently, the grafts might have failed to

receive the necessary stimulation to pre-

vent graft resorption. In fact, the resorption

of autogenous bone graft has not only been

reported in a sinus lifting model but has

also been described in studies that explored

ridge augmentation and bone defect’s re-

storation (Chenug et al. 1994; Roccuzzo

et al. 2007).

In our study, both group A and B showed

the convex augmented space, which sug-

gested that the grafted material can with-

stand sinus air pressure and maintain the

augmented space. The statistical results

showed that both groups maintained their

augmented height throughout the observa-

tion period (P40.05). OsteoBonet alone

achieved increased bone area while the

addition of bMSCs to this material demon-

strated the most promising results.

Throughout the experiment, both these

groups showed increased bone area and

maturation of newly formed bone. Lamel-

lar bone structure in histological sections

was observed 8 weeks after surgery.

In these experiments, no animals

showed signs of infection after surgery.

Histologically, newly formed bone was

found growing into the pores of the mate-

rial from the host bone and had a direct

bond with the graft without obvious in-

flammatory cell infiltration. Furthermore,

cells were found spreading along the mate-

rial surface after being cultured in vitro.

These results suggest that OsteoBonet has

a good biocompatibility and can facilitate

bMSCs adhesion onto its surface. Similar

results have also been observed by Xu et al.

(2004) when using deproteinized bone. Ac-

cording to Fleming et al. (2000), high

calcium phosphate concentrations appear

to improve the integration of implants into

host bone. One possible mechanism is that

the high concentrations of calcium and

phosphate ions may initiate biomineraliza-

tion or may influence osteoblast differen-

tiation in the cells found in adjacent tissues

(Damien et al. 1994). Moreover, silica ion

in the material also affords biocompatibil-

ity. When comparing the effects of different

calcium phosphate particles on the growth

of osteoblasts in vitro, Sun et al. (1997)

found that, under the same experimental

conditions, cell population, the concentra-

tions of TGF-b and ALP cultured on the

silica surface were similar with those on

normal plates. Knabe et al. (2005) found

that novel glass ceramics with silica sup-

ported cellular proliferation as well as the

expression of osteogenic markers as much

as, or better than, tri-calcium-phosphate.

Hard tissue engineering may potentially

provide a better alternative than currently

available bone grafts (Petrovic et al. 2006).

To this end, we used a new inorganic

material (i.e. OsteoBonet) as a scaffold

and bMSCs as seed cells to form a compo-

site of grafted material for maxillary sinus

floor elevation. We found that new bone

was formed mainly in areas close to the

parent bone in both group A and group B 2

weeks after transplantation. From 4 weeks

to 8 weeks, the bone area in group A

(bMSCs/material) increased significantly

from 23.47� 3% to 35.36� 10.57%

(Po0.05). However, the increase in bone

area in group B (material alone) at any time

was not significant. In comparison with

the autogenous group, the bone area in the

bMSCs/material group was much higher at

8 weeks (Po0.05); however, the difference

of bone volume between the material group

and autogenous bone was not significant.

At 8 weeks, the mean bone area in group A

(bMSCs/material) was 30% more than

that in the material-alone group, but differ-

ence of bone area between the bMSCs/

material group and the material-alone

group was not significant. This might be

due to the large variability associated with

the limited number of animals used in this

study.

OsteoBonet has pores with a suitable

diameter that is good for cell adhesion and

growth (Sun et al. 1997). We observed more

new bone formation in the bMSCs/mate-

rial group than in group material after

transplantation. This result concurred

with the results from the fluorescent label-

ing experiments. From the fluorescent

images taken, all three fluorescent colors

were intense and were found extensively in

the bMSCs/material site, which indicated

that the mineralization of newly formed

bone occurred after 2 weeks, while the

material-alone group only demonstrated

intense fluorescence at around week 8.

This suggests that bMSCs have contributed

to the increased bone area as well as en-

hanced mineralization. In the material-

alone group, the results suggested that

new bone formation depended on the os-

teoconductive property of the material, as

the newly formed bone was observed

mainly at the periphery of the parent

bone. When bMSCs were used along with

the OsteoBonet, these cells filtrated into

the pores of the material and more bone

formed in the inner area.

In this study, we simply focused on new

bone formation and the change of augmen-

ted height to reflect the general effects of

sinus elevation. According to the data, the

rate of resorption seemed slow. In some

areas, however, we found osteoclasts on

the surface of the interface between newly

formed bone and the material (data not

shown). This suggests that osteoclastic

resorption might be one of the methods

through which resorption occurred. Ac-

cording to Cornell (1999), calcium phos-

phate-rich surface layers can stimulate

osteoclastic resorption. In future studies,

the degradation of OsteoBonet will be

explored using quantitative methods to

calculate the degradation rate, and using

histochemical staining to assess osteoclasts

activity.

Besides the augmented height and bone

area, the quality of newly formed bone is

also an important factor affecting the sta-

bility of implants. At 2 weeks, the new

bone was composed of irregular trabeculea

with large bone lacuna in both group A



(bMSCs/material) and group B (material-

alone). However, 8 weeks after surgery,

new bone was more mature (as evidence

by lamellar structure formation and the

presence of bone marrow therein) in group

A (bMSCs/material) (data not shown).

Schmelzeisen et al. (2003) used a tissue-

engineered bone complex of periosteum-

derived osteoblasts with a polymer to lift

patients’ sinus and found lamellar bone

formation within 4 months. They sug-

gested that lamellar bone could allow for

reliable implantation. Bone marrow sug-

gested the maturation and remodeling of

newly formed bone, but little bone in large

areas of fatty marrow obviously would

decrease the stability of implants. Thus,

how new bone structure, including the

presence of bone marrow, would affect

the real mechanical stability of engineered

bone for implantation must be addressed in

future studies.

In summary, the combination of bMSCs

and OsteoBonet material can be used suc-

cessfully as a bone graft for maxillary sinus

lift in rabbits when compared with the use

of autogenous bone and blood clot. Because

the final goal of elevating maxillary sinus is

for dental implant placement and occlusion

restoration, further experiments using lar-

ger animal models with dental implants, as

well as a longer observation time, will be

needed in order to provide more clinically

relevant data.

Acknowledgements: This work was

supported by National Natural Science

Foundation of China 30400502,

30772431. Science and Technology

Commission of Shanghai Municipality

04dz05601, 05DJ14006, 055407034,

07DZ22007, Shanghai Rising-star

Program 05QMX1426. Shanghai

Education Committee 03BC39,

04YQHB081, Y0203, 07SG19 and National

High Technology and Development

Program of China 2002AA205011.

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