Dolly MushaharyCuie WenJerald M. KumarRagamouni SravanthiPeter HodgsonGopal PandeYuncang Li

Strontium content and collagen-Icoating of Magnesium–Zirconia–Strontium implants influenceosteogenesis and bone resorption

Authors’ affiliations:Dolly Mushahary, Peter Hodgson, Yuncang Li,Institute for Frontier Materials, Deakin University,Geelong, Vic, AustraliaCuie Wen, Faculty of Engineering and IndustrialSciences, Swinburne University of Technology,Hawthorn, Vic, AustraliaJerald M. Kumar, Ragamouni Sravanthi, GopalPande, CSIR-Centre for Cellular and MolecularBiology, Hyderabad, India

Corresponding author:Yuncang Li, PhDInstitute for Frontier Materials, Deakin UniversityWaurn Ponds, GeelongVictoria 3217, AustraliaTel.: +61 3 52272168Fax: +61 3 522 711 03e-mail: yuncang.li@deakin.edu.au

Key words: bone remodelling, calcification, gene expression, matrix mineralisation, osteo-

clasts

Abstract

Objective: Our objective was to study the role of Collagen type-I (Col-I) coating on Magnesium–

Zirconia (Mg–Zr) alloys, containing different quantities of Strontium (Sr), in enhancing the in vitro

bioactivity and in vivo bone-forming and mineralisation properties of the implants.

Materials and methods: MC3T3-E1 osteoblast cell line was used to analyse the in vitro properties

of Col-I coated and uncoated alloys. Cell viability analysis was performed by MTT assay; cell

attachment on alloy surfaces was studied by scanning electron microscopy (SEM); and gene

profiling of bone-specific markers in cells plated on uncoated alloys was performed by Quantitative

RT-PCR. In vivo studies were performed by implanting 2-mm-sized cylindrical pins of uncoated and

coated alloys in male New Zealand white rabbits (n = 33). Bone formation and mineralisation was

studied by Dual Energy X-ray Absorptiometry (DXA) and histological analysis at one and three

months post-implantation.

Results: Our results clearly showed that Sr content and Col-I coating of Mg–Zr–Sr alloys

significantly improved their bone inducing activity in vitro and in vivo. Osteoblasts on coated alloys

showed better viability and surface binding than those on uncoated alloys. Sr inclusion in the

alloys enhanced their bone-specific gene expression. The in vivo activity of implants with higher Sr

and Col-I coating was superior to uncoated and other coated alloys as they showed faster bone

induction and higher mineral content in the newly formed bone.

Conclusion: Our results indicate that bone-forming and mineralising activity of Mg–Zr–Sr implants

can be significantly improved by controlling their Sr content and coating their surface with Col-I.

Magnesium (Mg) alloys are attractive for sev-

eral biomedical applications, ranging from

dental prostheses (Sehlke et al. 2013) to car-

diovascular implants (Heublein et al. 2003).

They exhibit mechanical properties that are

similar to human bone tissue such as similar

material density and compressive strength

(Staiger et al. 2006). In addition, biocompati-

ble Mg alloys are non-toxic and biodegradable

– their in vivo biodegradation progressively

stimulates their own activity and minimises

their re-accumulation and toxicity (Staiger

et al. 2006).

The role of other metals in Mg alloys has

been shown to influence their bone-forming

properties (Witte et al. 2005; Zhang et al.

2009; Gu et al. 2012; Yang et al. 2012).

Among other metals, the use of strontium

(Sr) in implants has become popular because

strontium ranelate (SrR) has been approved

for the treatment for osteoporosis, under the

trade name of Protelos (Meunier et al. 2004;

Reginster et al. 2008). Our earlier studies

have demonstrated improved in vivo biocom-

patibility, osseointegration and bone remodel-

ling by modulating the contents of Sr and

Zirconia (Zr) in Mg–Zr–Sr implants (Mushah-

ary et al. 2013; Sravanthi et al. 2013). Other

studies have also indicated the role of Sr con-

tent in implants for better bone formation

(Grynpas & Marie 1990; Canalis et al. 1996),

improved bone resorption (Buehler et al.

2001; Marie 2007), higher bone collagen syn-

thesis (Canalis et al. 1996), more precipita-

tion of hydroxyapatite (Cheung et al. 2005;

Ni et al. 2006) and increased hardness of tra-

becular and cortical bones (Cattani-Lorente

et al. 2013). Sr has been shown to have some

Date:Accepted 16 September 2014

To cite this article:Mushahary D, Wen C, Kumar JM, Sravanthi R, Hodgson P,Pande G, Li Y. Strontium content and collagen-I coating ofMagnesium–Zirconia–Strontium implants influenceosteogenesis and bone resorption.Clin. Oral Impl. Res. 00, 2014, 1–10.doi: 10.1111/clr.12511

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1

negative effects on bone formation also such

as defective or abnormal mineralisation of

the bone matrix (Schrooten et al. 1998a,

2003; D’Haese et al. 2000), hence the use of

Sr in bone implants needs to be re-evaluated.

The use of implants with bioactive coatings,

such as RGD peptides or Col-I, also stimu-

lates osteoinduction (Schaffner et al. 1999).

These coatings enhance the interaction

between the implant surface and extracellu-

lar matrix (ECM) at the implant site, thereby

increasing mineralisation of the pre-existing

bone and making it more rigid and load-bear-

ing.

In this study, we have analysed the in vitro

and in vivo properties of Col-I-coated Mg–Zr

alloys containing high (5 wt%) or low (2 wt

%) quantities of Sr. We have evaluated the

dose and time-dependent influence of Sr

quantity and Col-I coating of the implants.

Our investigation indicated that Sr content of

implants influenced bone-specific gene

expression in vitro and implant bone apposi-

tion during in vivo bone formation; Col-I

coating of implants enhanced the in vivo

mineral deposition in newly formed and pre-

existing bones.

Material and methods

Alloy preparation

The Mg alloy discs and cylinders used for

in vitro and in vivo experiments, respec-

tively, were fabricated by casting from melts

of commercial pure Mg (99.9%), master

Mg-30Sr and Mg-33Zr alloys with designated

compositions under pure argon atmosphere

with a steel crucible. The cylindrical in vitro

implant samples of 5 mm diameter and

10 mm height were cut from the ingot by

electrical discharge machining (EDM) for

mechanical characterisation and microstruc-

ture observation. The in vivo implants of

2 mm diameter and 4 mm height were pol-

ished using 600-grit silicon carbide (SiC)

papers followed by degreasing ultrasonically

with acetone, ethanol and distilled water and

air-dried. For surgical use, the in vivo

implants were ultrasonicated in 2% chro-

mium oxide for 20–30 min followed by wash-

ing with acetone, ethanol and distilled water

for 10 min each, air-dried and UV sterilized.

Table 1 lists the composition of the three

Mg-based alloys. The mechanical and physi-

cochemical properties of these alloys have

been discussed earlier (Li et al. 2012; Mu-

shahary et al. 2013). In this study, three types

of Mg-based alloys were used to illustrate the

role of increasing concentrations of Sr in

bone mineralisation: Mg–5Zr, Mg–Zr–2Sr and

Mg–2SZr–5Sr, which will be henceforth

referred to as no-Sr, low-Sr and high-Sr,

respectively.

Collagen extraction and dip coating

Col-I from rat tail tendons was extracted

according to the protocol described by Rajan

et al. (2007). Dip coating was performed

according to the procedure described by Geiß-

ler et al. (2000). Lyophilised collagen was

then dissolved in 0.25 N acetic acid and was

used for dip coating at a concentration of

5 lg/ll. Alloy dip coating was conducted

using a Single Dip Coater (Model No SDC

2007C, Apex Instruments Co., Kolkata, West

Bengal, India) with SDC Software provided by

the instrument suppliers.

Cell cytotoxicity

Mouse osteoblast cell line MC3T3-E1

(ATCC, USA) was used to check the cytotox-

icity of Mg–Zr–Sr alloys using MTT [3-(4, 5-

dimethylthiazol-2-yl)-2, 5-diphenyl tetrazo-

lium bromide] assay based on the indirect

method by standard ISO 10993-5 protocol

(ANSI/AAMI/ISO 1999). The alloy extracts

were prepared by incubating the substrates in

complete medium consisting of alpha-MEM

supplemented with 10% foetal calf serum

(FCS), 100 U/ml penicillin and 100 lg/ml

streptomycin at 37°C in a humidified atmo

sphere of 95% air and 5% CO2 for 24 h with

surface area of alloy to media ratio of

0.6 cm2/ml and sterilized using 0.22 l filter

(Merck Millipore, Darmstadt, Germany).

Cell culture for MTT assay and gene

expression studies was performed according

to Sista et al. (2013). The morphology of the

cells for the two time points was observed

with Scanning Electron Microscopy (SEM)

(3400N Hitachi, Ibaraki, Japan). Due to early

degradable nature of Mg alloys under in vitro

conditions, we performed the gene expression

studies by indirect method of cell culture.

Cells for gene expression studies were

allowed initial adhesion of 4 h, after which

the complete media was replaced by alloy

extracts without disturbing the cells. They

were then incubated in standard conditions

for 24 h (1 d) and 7 days. The alloy extract

was supplemented with conditioned media at

an interval of 2 days for 7 days culture to

maintain the media volume.

Quantitative RT-PCR

Total RNA from each culture flask was pre-

pared using Qiagen RNA Plus kit (QIAGEN,

Valencia, CA, USA) as per the manufacturer’s

instructions. The total RNA from two sepa-

rate experiments at 2 specified time points

was isolated for preparing cDNA. The iso-

Table 1. Composition of the alloying elements in Mg implants

Alloys

Chemical compositions (Wt%)

Zr Sr Al Si Fe Mn Mg

No-Sr 4.88 – 0.02 0.01 0.01 0.01 BalanceLow-Sr 0.92 1.82 0.04 0.02 0.02 0.01 BalanceHigh-Sr 1.89 4.8 0.03 0.02 0.01 0.01 Balance

Table 2. Forward and reverse primers of genes used for quantitative RT-PCR experiments.

Gene Primers (50 – 30) Product length (bp)

GAPDH Fwd – AGC GAG ACC CCA CTA ACA TCA 21Rev – CTT TTG GCT CCA CCC TTC AAG T 22

ALP Fwd – ACC CGG CTG GAG ATG GAC AAA T 22Rev – TTC ACG CCA CAC AAG TAG GCA 21

Collagen Type-I Fwd – CTC CTG ACG CAT GGC CAA GAA 21Rev – TCA AGC ATA CCT CGG GTT TCC A 22

Osteopontin Fwd – TGA TTC TGG CAG CTC AGA GGA 21Rev – CAT TCT GTG GCG CAA GGA GAT T 22

Osteonectin Fwd – ATG TCC TGG TCA CCT TGT ACG A 22Rev – TCC AGG CGC TTC TCA TTC TCA T 22

Bone sialoprotein Fwd – ACC GGC CAC GCT ACT TTC TTT A 22Rev – GGA ACT ATC GCC GTC TCC ATT T 22

Osteocalcin Fwd – AGC AGG AGG GCA ATA AGG TAG T 22Rev – TCG TCA CAA GCA GGG TTA AGC 21

BMP2 Fwd – TGG AAT GAC TGG ATC GTG GCA C 22Rev – ACA TGG AGA TTG CGC TGA GCT C 22

Fibronectin Fwd – TGC AGT GGC TGA AGT CGC AAG G 22Rev – GGG CTC CCC GTT TGA ATT GCC A 22

2 | Clin. Oral Impl. Res. 0, 2014 / 1–10 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

lated RNA was quantified using a nanodrop

spectrophotometer (Thermo Fisher Scientific

Inc., Waltham, MA, USA). The quantified

RNA was checked on 0.8% agarose gel elec-

trophoresis for its purity. cDNA synthesis

and quantitative RT-PCRs were performed as

described by Sista et al. (2013). Table 2 shows

the forward and reverse primers of genes used

for real-time PCRs.

Animal surgery

In each experiment containing 33 animals,

five groups were made according to the time

period of implantation and the alloy implants

used: one group for control animals of one

month surgery period (n = 3), second group for

control animals of three months surgery per-

iod (n = 3), third group for coated implants of

one month implantation period (n = 9), fourth

group for coated implants of three months

implantation period (n = 9) and fifth group for

uncoated implants of three months implanta-

tion period (n = 9). Triplicate animals were

used for the control and each of the alloys.

Control animals had a hole drilled the same

size of the implant without any alloy. Surgical

procedures were performed as per the ethical

clearance performed by the institutional ani-

mal ethics committee of Deakin University

and CSIR-CCMB. Animal surgery was per-

formed on left hind leg and post-surgery ani-

mal monitoring was performed according to

the protocol described earlier (Mushahary

et al. 2013). Appropriate measures were taken

to avoid any kind of pain and discomfort dur-

ing and after surgery of the experimental ani-

mals. After the specified monitoring period,

the implanted femur was excised, cleaned and

washed in phosphate buffered saline (PBS) and

stored in 10% buffered formalin for histologi-

cal experiments.

Bone embedding and histology

The animals were euthanised and the excised

bone samples were embedded in methyl-

methacrylate (MMA) as described earlier (Er-

ben 1997; Mushahary et al. 2013). Mayer’s

Haematoxylin and Eosin (HE) staining (Luna

1968) and Tartrate Resistant Acid Phospha-

tase (TRAP) staining was conducted to study

the in vivo bone cell adhesion and matrix for-

mation at the peri-implant bone interface and

to assess bone turnover, respectively. TRAP

staining was performed by incubating the

slides in 50 : 0.5 proportions of Buffer I

[Sodium Acetate Anhydrous, L-(+) Tartaric

acid and Glacial Acetic acid] and Buffer II

(Naphthol AS-BI Phosphate and Ethylene

Glycol Mono-ethyl Ether) for 45 min at 37°C

followed by 50 : 1 : 1 proportions of Buffer I,

Buffer III (Sodium Nitrite) and Buffer IV

(Pararosaniline Hydrochloride and 2N Hydro-

chloric acid) for 5 min at room temperature.

The sections were counterstained with Hae-

matoxylin and then put into 0.05% ammonia

water followed by dehydration in alcohol.

Slides were cleared in xylene and mounted in

Permount� (Thermo Fisher Scientific Inc.,

Waltham, MA, USA). The new bone miner-

alisation was studied by Masson’s Trichrome

according to the manufacturer’s protocol

(Sigma Aldrich, St. Louis, MO, USA) and

confirmation of the mineralisation was made

by calcium staining of bone sections by Aliz-

arin Red (Nagy et al. 2003).

Bone mineral and density of coated alloys

The Dual Energy X-Ray Absorptiometry

(DXA) was performed to measure the Bone

Mineral Content (BMC) and Bone Mineral

Density (BMD), around the implant area, of

live animals, one and three months after

implantation in a Hologic Discovery QDR�

Series (Bedford, MA, USA) to record the min-

eralisation and density of the bone around

the implant site. The mineral concentration

and density of the region was calculated

using the QDR software provided by the

instrument suppliers.

Statistics

Statistical evaluation of data obtained from

three different animals per experiment for

each alloy and controls was used for the

determination of BMC and BMD of bones in

the implanted area. Data from three different

experiments were used for statistical evalua-

tion of cell viability. Two-way analysis of var-

iance (ANOVA) was performed using MiniTab

software. Standard errors in the data points

were determined at 95% confidence level. P-

values of less than 0.05 (<0.05) were consid-

ered statistically significant. Data presented

in the graphs or tables are mean values�SD.

Results

Cell viability, cell adhesion and mineralisation

The MC3T3-E1 osteoblasts showed good

compatibility and viability with the Col-I-

coated Mg–Zr–Sr alloys. Fig. 1 shows the

cell viability ratio with the uncoated and

coated-alloy extracts and Fig. 2 depicts the

SEM images of morphology and mineralisa-

tion of osteoblasts on the uncoated and

coated-alloy surfaces. The osteoblasts exhib

Fig. 1. Cell viability ratio of MC3T3-E1 osteoblasts cul-

tured with alloy extracts of bare and col-I-coated Mg–

Zr–Sr alloys incubated for 24 h. ***P < 0.001.

(a) (b) (c)

(d) (e) (f)

Fig. 2. Morphology of MC3T3-E1 osteoblasts on bare and coated Mg–Zr-based alloy surfaces after 24 h of incuba-

tion. Panels a–c show cells on the surfaces of bare –no-Sr, –low-Sr and –high-Sr alloy surfaces, respectively, and

panels d–e on coated –no-Sr, –low-Sr and –high-Sr alloy surfaces, respectively.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 3 | Clin. Oral Impl. Res. 0, 2014 / 1–10

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

ited cell death after incubation for 24 h on

the uncoated surface of no-Sr alloy. How-

ever, after coating the alloys with Col-I and

when cultured under similar conditions,

stronger cell adhesion demonstrating exten-

sive network and mineral deposition was

observed.

In vitro expression of bone markers

The comparative gene expression profiles of

bone-related markers in osteoblasts, after

their growth for 1 or 7 days in different

uncoated-alloy extracts, were measured by

RT-PCR (Fig. 3). Expression of all genes was

clearly detectable in cells after 7 days of

growth in all the alloy extracts; however, it

was higher in cells grown in extracts of

Mg–2Zr–5Sr as compared to those grown in

extracts of Mg–Zr–2Sr. This indicated that

higher Sr-containing alloys had better gene

inducing ability directed towards bone dif-

ferentiation. Interestingly, BMP-2 gene

expression was detectable at 24 h and

7 days but its levels were not significantly

different among the alloys at both time

points.

In vivo bone inducing activity

Fig. 4 shows HE stained images of the newly

formed bone around the Mg implants, after 1

and 3-months implantation. In the coated

implants, initiation of bone formation could

be seen early after 1-month implantation

(Panels a–c), which gives rise to mature and

fully developed trabecular bone after

3 months (Panels d–f). Very little or no dis-

continuity between the cement line matrix

and the newly formed bone could be seen for

coated Sr-containing implants, whereas a

large gap could be seen for coated no-Sr

implant. The uncoated alloys also showed

good amount of bone formation by Sr-con-

taining implants, after 3-months implantation

(Panels g–h). The TRAP-positive cells were

minimal in the sections of coated implants

(Fig. 5, panels a–f). However, the number of

TRAP stained cells was seen to be higher in

the bone sections of uncoated implants, espe-

cially for high-Sr alloy, suggesting excessive

bone turnover (Fig. 5, panel i). This difference

in staining, between the alloys containing

low and high-Sr and between the uncoated

and coated implants, indicates the role of Sr

concentration and Col-I coating in bone

induction and turnover by Mg–Zr–Sr alloys.

Implant-induced bone mineralisation

Fig. 6 shows Masson’s Trichrome staining,

demonstrating coated (panels a–f) and

uncoated (panels g–i) peri-implants. Homoge

neous mineral deposition on the new bone

could be seen in all the implant types, with

superior bone mineral in coated high-Sr

implant, after both 1 month and 3 months

(panels c and f) implantation. Interrupted and

heterogeneous bone mineral deposition was

seen by the uncoated implants 3-month post-

surgery (Panels g–i). Irrespective of the Sr

concentration in uncoated alloys, bone min-

eralisation could be seen in all the implant

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Fig. 3. The expression of genes related to bone-specific markers, proteins related to bone extracellular matrix and

proteins of non-collagenous bone matrix after 24 h (1d) and 7 days (7d) of incubation in the respective coated-alloy

extracts.

4 | Clin. Oral Impl. Res. 0, 2014 / 1–10 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

types. However, the time- and coating-depen-

dent bone mineral deposition by the coated

implants could have been influenced due to

controlled release of Sr ions by Col-I coating

in the vicinity of implant area. This result

matches the Alizarin Red staining of coated

implants (Fig. 7) in which, the coated high-Sr

alloy showed mature bone formation only

after 1-month implantation, with high-cal-

cium content after 3 months. Coated low-Sr

implant showed partial Alizarin Red staining

for the same 3-months period of implanta-

tion, whereas coated no-Sr implant displayed

very little staining. The calcium content of

newly formed bone indicates its extent of

maturity, and the bone response observed in

the sections of coated implants suggests the

influence of Sr content in bone formation

and its degree of calcification.

Bone density and mineral concentration

The BMC and BMD of the implant sites give

us an idea about bone healing from the min-

eral content and density of the newly formed

bone. As seen in Fig. 8, there is a significant

increase of BMC and BMD in the coated

high-Sr implant site, compared with the

uncoated and coated alloys having no-Sr or

lower concentration of Sr, in both the

implant periods (1 month and 3 months).

These results agree with our histology data

which confirms that Col-I-coated high-Sr

implant is superior to other uncoated and

coated alloys in enhancing bone mineral for-

mation in rabbit femur.

Statistics

A two-way ANOVA test showed a very signifi-

cant effect of surface modification, F (1,

(a)

(d)

(b) (c)

(e) (f)

(g) (h) (i)

Fig. 4. Haematoxylin and Eosin staining of bone sections implanted with Col-I coated and uncoated Mg–Zr-based alloys after 1-month and 3-month post-implantation. Panels

a–c: Col-I-coated 1-month study, Panels d–f: Col-I-coated 3-month study, Panels g–i: Uncoated 3-month study.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 5 | Clin. Oral Impl. Res. 0, 2014 / 1–10

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

12) = 82.73, P < 0.001, and implant types, F

(2, 12) = 40.32, P < 0.001, on cell viability.

There was also a very significant interaction,

F (2, 12) = 15.67, P < 0.001, of these two

variables on the cell viability. Similar analy-

sis was run on a sample of 24 animal mod-

els, grouped each for BMC and BMD

analysis, to examine the effect of period of

implantation and type of implants on femur

BMC and BMD. Significant effect of implant

types, F (3, 16)= 5.87, P = 0.007, could be

seen on BMD response. However, there was

no significant interaction of the above two

variables on BMC [F (3, 16) = 1.00,

P = 0.418] and BMD [F (3, 16) = 0.93,

P = 0.447].

Discussion

There has been significant progress in devel-

oping biodegradable alloys that exhibit good

biocompatibility and osteoinducing proper-

ties (Gu et al. 2014; Henderson et al. 2014).

Mg-based alloys have proven to be useful in

this direction because of their properties

mentioned above (see references in Introduc-

tion). However, further optimisation of their

constitution and modifications of their sur-

face characteristics are necessary to make

them biologically and physicochemically

more suitable for medical applications.

Extending upon some of our earlier results

on Mg-based alloys (Li et al. 2012; Mushah-

ary et al. 2013; Sravanthi et al. 2013), we

have enumerated the role of Sr concentration

and Col-I coating in Mg–Zr–Sr alloys with

reference to their in vitro and in vivo bone

inducing potential.

Our results in Figs 1 and 2 showed that high-

Sr concentration in Mg-based alloys is associ-

ated with superior viability and proliferation of

(a) (b)

(d) (e) (f)

(g) (h) (i)

(c)

Fig. 5. Tartrate resistant acid phosphatase staining of osteoclasts for bone sections implanted with Col-I coated and uncoated Mg–Zr-based alloys after 1-month and 3-months

implantation. Panels a–c: Col-I-coated 1-month study, Panels d–f: Col-I-coated 3-months study, Panels g–i: Uncoated 3-months study.

6 | Clin. Oral Impl. Res. 0, 2014 / 1–10 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

osteoblasts on their surfaces. These alloys

also induced higher expression of genes

related to bone markers (Fig. 3). The level of

bone formation and its maturation was found

to be affected by time up to three months, as

seen from the histology and DXA results

(Fig. 8). As the collagenous matrix of newly

formed bone is depicted by Masson’s Tri-

chrome staining, our results indicate that

increased Sr concentration in Mg alloys influ-

ences the collagenous matrix formation by

osteoblasts, without inducing any negative

effects in long-term matrix mineralisation, as

also stated by Barbara et al. (2004). These

results are also consistent with previous

studies showing that Sr increases trabecular

bone formation in vivo in rats, mice and

humans (Marie 2003). From the histology

results, no defects in bone apposition and cal-

cification were observed. This homogeneity

could be because all the existing bone around

the peri-implant area consisted of new bones,

formed during the period of implantation.

Also, there is a decreased expression of osteo-

clast markers in the bone sections of high-Sr-

containing implants, indicating the inhibi-

tory effect brought upon by Sr concentration

on osteoclast resorption, without any cyto-

toxic effects. Although the precise mecha-

nism of the role of Sr concentration on

osteogenesis and bone turnover is not fully

understood, the increased bone formation and

its suppressed turnover, within the specified

implant period, suggests that Sr is dose

dependently taken up and distributed by the

osteoblasts in newly formed bone tissue.

The Sr ions released from the degrading

implant possibly affects the osteocytes,

which in turn controls the activity of osteo-

blasts and osteoclasts (Tan et al. 2014). The

mechanical pressure applied by the newly

forming bone matrix is sensed by osteocytes.

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Fig. 6. Masson’s Trichrome staining of bone sections implanted with Col-I coated and uncoated Mg–Zr-based alloys 1-month and 3-months post-implantation. Panels a–c: Col-

I-coated 1-month study, Panels d–f: Col-I-coated 3-months study, Panels g–i: Uncoated 3-months study. The blue stain indicates collagen matrix of mineralised bone.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 7 | Clin. Oral Impl. Res. 0, 2014 / 1–10

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

Less activity in the surrounding peri-implant

area activates the osteoclasts for bone resorp-

tion or inhibits the osteoblasts for bone for-

mation. These phenomena are the response

to mechanical stimuli, and any factor that

affects these stimuli can affect bone forma-

tion and/or resorption. Sr can replace this

mechanical stimuli, as osteocytes devoid of

any external stimuli enhance the osteoclast

bone resorption in vivo, as seen in our

uncoated Sr-containing implant sections and

reported by others (Xiong et al. 2001; Tats-

umi et al. 2007). Considering the site of bone

resorption and the position of osteoclasts, the

applied pressure signals the osteoclasts to

reduce their activity in the presence of cer-

tain concentration of Sr, which in our study

has been shown to be 5 wt%. The activated

osteocytes also produce osteoblast-stimulat-

ing factors, including BMP-2, for bone forma-

tion. Also, the action of Sr on bone

resorption can be associated with a Ca-like

effect on bone, wherein high concentrations

of Ca have been shown to inhibit osteoclast

activity in vitro (Zaidi et al. 1999) and in

vivo (Malgaroli et al. 1989).

The enhanced osteogenesis can also be

associated to Col-I coating. Availability of

coating on the Mg alloy surface leads to

gradual release of degrading Sr ions upon

implantation. The slow release of ions not

only promotes osteoblast proliferation, but

also facilitates the precipitation of apatite,

thus increasing the mechanical strength of

the bone-implant interface (Cheung et al.

2005; Ni et al. 2006). Consistent with this

interpretation, Barbara et al. (2004) also

found that Sr uptake is directly proportional

to Mg uptake and free Mg ions act as stim-

ulators of osteoblast activity (Mushahary

et al. 2013). In our study, the gradual

release of Sr ions due to Col-I coating

(a) (b) (c)

(d) (e) (f)

Fig. 7. Alizarin Red staining of bone sections implanted with Col-I-coated Mg–Zr-based alloys after 1-month and 3-months implantation. Panels a–c: Col-I-coated 1-month

study, Panels d–f: Col-I-coated 3-months study. The red stain indicates accumulation of calcium in mineralised bone.

Fig. 8. Quantitative in vivo mineralisation of the peri-implant area by evaluation of femur bone mineral content

and bone mineral density of experimental animals implanted with Col-I-coated Mg–Zr-based alloys after 1-month

and 3-months implantation. *P < 0.05.

8 | Clin. Oral Impl. Res. 0, 2014 / 1–10 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Mushahary et al �Collagen coating influence bone morphogenesis and resorption

creates availability of free Mg ions for

longer period throughout the implantation.

This could have contributed to superior

osteoblast activity and a subsequent increase

in ECM quantity in the Sr-containing peri-

implant bone area. Hence, Col-I acts as a

connecting factor between the alloy surface

and the extent of availability of degrading

ions to the peri-implant area required for

bone ECM quality and quantity.

Our findings, along with the previous

report (Mushahary et al. 2013), raise the pos-

sibility that high-Sr content (5 wt%) in Mg

alloys may have beneficial effects on osteo-

genesis, with decreased bone resorption,

while maintaining enhanced new bone min-

eralisation, when coated with Col-I. How-

ever, it affects not only the process of new

bone formation but also the bone mineral

deposition, as seen from the histology and

BMC/BMD results of DXA radiography in

our study. However, further studies need to

be carried out to understand the precise

impact of Sr concentration on bone minerali-

sation. It would be valuable to conduct future

work to estimate the precise amount of new

bone that is formed around the implant. Mea-

suring the deposition of Sr ions in the newly

formed bone will provide us with a more

accurate knowledge of Sr incorporation in the

implant site from biodegrading Mg implants.

Gene expression for osteoblasts grown in

coated-alloy extracts was not quantified due

to the potential effect of coated Col-I in the

growth of cells. Nonetheless, our study helps

in understanding the basic optimum elemen-

tal constituents required in Mg alloys for

their application as orthopaedic implants.

Conclusion

Our work provides experimental evidence

that Mg-based alloys containing high-Sr (5 wt

%) and coated with Col-I, enhances the rate

of osteogenesis and reduces bone resorption

over short-term implantation, resulting in

increased bone mass. This gives rise to

increased trabecular bone volume without

altering mineralisation process of the newly

formed bone. Such alloys can help in preven-

tion of bone loss and increase bone mass and

maturity. This work confirms that prolonged

exposure of Sr to the implant site results in

better bone formation and lower bone turn-

over, without any deleterious effects of long-

term Sr exposure on bone mineralisation. We

also confirm the effect of Col-I-coated high-

Sr-containing Mg-based alloys in bone resorp-

tion, by altering the in vivo osteoclast proper-

ties. Our data describe a positive effect of

Col-I-coated high-Sr-containing Mg alloy

(Mg–2Zr–5Sr) on osteoblasts and mineral

deposition and negative effect on osteoclasts

and bone resorption.

Acknowledgements: The authors

acknowledge the financial support from the

Department of Industry, Innovation, Science,

Research and Tertiary Education (DIISRTE),

Australia, through the Australia-India

Strategic Research Fund (AISRF, BF030031 to

PH and CW) and the Department of

Biotechnology (DBT), Government of India,

Australia India Biotechnology Fund (AIBF,

ICD/55/11, GAP0311 to GP). PH

acknowledges the support from the

Australian Research Council (ARC) through

Australian Laureate Fellowships. The authors

acknowledge the technical assistance of staff

members of CSIR-CCMB Animal House. DM

acknowledges Dr. Jane Allardyce for

manuscript editing.

Conflict of interest

The authors state that they have no conflict

of interests.

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