Hydroxyapatite as a Carrier for Bone
Morphogenetic Protein

Ramin Rohanizadeh*
Kimberly Chung

Bone morphogenetic proteins (BMPs) can induce the formation of new bone in numerous
orthopedic and dental applications in which loss of bone is the main issue. The combination
of BMP with a biomaterial that can carry and deliver proteins has been demonstrated to
maximize the therapeutic effects of BMPs. However, no ideal candidate with optimal
characteristics as a carrier has emerged for clinical use of BMPs. Hydroxyapatite (HA) is a
potential BMP carrier with its osteoconductive properties and desirable characteristics as a
bone graft biomaterial. In this study, 3 different methods to load BMP into HA materials were
characterized and compared based on the BMP uptake and release profile. BMP was loaded
into HA in 3 ways: (1) incorporation of BMP during HA precipitation, (2) HA immersion in
BMP solution, and (3) BMP incorporation during dicalcium phosphate dihydrate (DCPD)
conversion to HA. The size of HA crystals decreased when BMP was loaded during HA
precipitation and HA immersion in BMP solution; however, it did not change when BMP was
loaded during DCPD-to-HA conversion. The highest BMP uptake was achieved using the
immersion method followed by HA precipitation, and the lowest via DCPD conversion. It is
interesting to note that BMP loading during HA precipitation resulted in sustained and
prolonged BMP release compared with the 2 other BMP loading methods. In conclusion, BMP
incorporation during HA precipitation revealed itself to be the best loading method.

Key Words: hydroxyapatite, bone morphogenetic protein, drug delivery, carrier

I

NTRODUCTION

A

lthough bone is one of the few

regenerative

tissues

in

the

human body, its regenerative

ability is limited.

1

Some bone

defects undergo incomplete

fracture healing (nonunion fractures), or the

defect size is beyond the body’s healing

capacity (critical size defects).

1–3

In such

cases, further intervention is required, typi-

cally of a surgical nature, to replace the

damaged bone with a bone graft material.

Osteoinductive

materials

such

as

bone

growth factors are able to lead and induce

bone formation, even in nonskeletal sites

(eg, muscle). Biomaterials used in orthopedic

applications, such as ceramics, metals, and

polymers, are only osteoconductive in nature

and generally are not able to induce bone
formation.

4

The combination of an osteo-

conductive biomaterial with osteoinductive

molecules overcomes the limitations of

synthetic bone graft biomaterials. One such

hybrid material involves incorporating bone
morphogenetic proteins (BMPs) into a bio-

degradable carrier. Once this hybrid material

is implanted, it will accelerate bone forma-

tion and regeneration in surrounding defec-

tive bone tissues.

BMPs are part of the transforming growth

factor-b cytokine family.

2

BMPs have a range

Advanced Drug Delivery Group, Faculty of Pharmacy,
University of Sydney, Sydney, Australia.
* Corresponding author, e-mail: ramin.rohanizadeh@
sydney.edu.au
DOI: 10.1563/AAID-JOI-D-10-00005

R

ESEARCH

Journal of Oral Implantology

659

of functions, including fetal organ develop-

ment and postnatal kidney, neuronal, and

bone development.

5

Of greatest interest are

its osteoinductive abilities, which allow BMP

signaling to induce postnatal bone forma-

tion in nonskeletal sites.

6,7

Of the many

isoforms, BMP-2 and -7 show the greatest

in vivo potential for osteogenesis.

8,9

They

recruit undifferentiated mesenchymal stem

cells to the defective site that differentiate

into osteoblasts, which form new bone

6,7

;

this is known as intramembranous bone

formation.

10

Another bone formation path-

way, endochondral formation, occurs when

chondroblasts (cartilage cells) are created

from stem cells, modulating the bone shape;

they are then calcified and replaced by

bone.

10

Both pathways can be induced by

BMPs and are highly dependent on the

properties of the BMP carrier and the isoform

of BMP.

10,11

The main interest behind using

BMP in dental and orthopedic applications is

its potential for reconstructing critical size

bone defects and nonunion fractures.

3

To

date, areas for which BMP have been studied

include spinal fusion, long bone trauma,

ligament reconstruction, and orthopedic,

craniomaxillofacial,

and

periodontal

dis-

ease.

3,12

Several BMP delivery systems are

available, ranging from the gene therapy

approach to local implantation and systemic

administration.

2

However, most research has

focused on localized delivery systems, par-

ticularly in search of the ideal BMP carrier.

The carrier for BMP should enhance the

activity of the protein by maintaining a

certain BMP concentration in the defective

area for a sufficient time to allow new bone

formation.

3,6,12–14

Good affinity should exist

between BMP and its carrier to maintain sus-

tained and prolonged BMP release. The carrier

should be easily sterilized and biodegradable

and should have no immunogenicity.

12,14

An

ideal carrier not only is a mechanism for drug

delivery but preferably should support bone

growth by having an appropriate porous

structure for cell infiltration and ingrowth.

Finally, the overall manipulation process that

includes loading BMP onto a carrier and its

release must retain the biological activity of

BMP.

6,11,12

A range of BMP carriers of an

organic and an inorganic nature have been

investigated.

2,13

These carriers have showed

varying levels of success, but the major

components of human bone—collagen and

hydroxyapatite—are preferred BMP carriers.

2

Hydroxyapatite

(HA),

Ca

10

(PO4)

6

(OH)

2

,

has been used as a carrier for antibiotics,

analgesics, and anticancer agents for the

skeletal system.

15,16

It is a preferred bioma-

terial in orthopedic and dental applications

because of its chemically similar structure

to the inorganic component of bone and

other hard tissues.

2,17

Although more soluble

phases of calcium phosphate exist (eg,

octacalcium phosphate, dicalcium phos-

phate dihydrate), HA has shown the greatest

potential in bone tissue engineering.

3

Syn-

thetic HA may be created through several

techniques and in a range of forms and

shapes, such as powder, blocks, discs, and

granules.

12

HA is capable of directly bonding

to bone, and emerging evidence suggests

osteoinductivity with HA materials of certain

pore sizes and characteristics.

18,19

BMP adsorption onto HA can enhance

interfacial strength and contact between

the HA implant and surrounding bone,

thereby promoting greater bone regenera-

tion around the implant than is seen with HA

alone.

3,17

On the other hand, incorporating

BMP into an HA porous carrier, even in small

doses, may increase the strength of the

porous carrier.

20,21

BMPs adsorb on HA

crystals via binding between functional

groups COO

2

, OH, and NH

2

of BMP and

the calcium site of HA.

22

One BMP molecule

may adsorb on HA, or up to 3 molecules may

adsorb cooperatively at once.

22

It has been

shown that the right side of BMP-2 is most

prone to adsorption, with maximal adsorp-

tion occurring in acidic conditions.

23

Acidic

Hydroxyapatite as a Carrier for BMP

660

Vol. XXXVII/No. Six/2011

conditions result in higher BMP/HA affinity

because below the isoelectric point of BMP

(pH 7.9), BMP is positively charged and HA

has a negative surface charge.

23

BMP adsorption is thought to be limited to

the macropores of HA, but there is a tendency

for a rapid initial burst release of BMP once a

BMP/HA composite is implanted in the

body.

23–25

Transient high BMP concentrations

in the bone defect can lead to adverse effects

by activating osteoclast cells, which in turn

resorb bone and promote inhibitory proteins

of BMP, thus limiting the osteogenic ability of

the implant.

24

Most studies have loaded BMP

to HA via coating of the surface of HA materials

by BMP. This can be achieved by immersion of

HA materials in a BMP solution because it can

be done under physiologic conditions, reduc-

ing the possibility of BMP denaturation. In

contrast, incorporating BMP into HA materials

results in BMP adsorption onto both macro-

pores and micro-pores of materials with

predominant attachment to calcium sites.

Hence, BMP incorporation into HA is thought

to prolong the retention profile because BMP

should be released as HA is dissolved.

13

Liu

et al

24

recently demonstrated that incorpora-

tion of BMP into biomimetic calcium phos-

phate revealed osteoinductive properties in

rats and was fivefold more efficacious in

osteoinductivity compared with implants sur-

face coated with BMP.

24

Many animal studies have been conduct-

ed on HA/BMP hybrid materials. These trials

used HA materials immersed for a time in a

BMP solution and convincingly demonstrated

HA as a suitable carrier for BMP.

3,7,17,25,26

Studies involving BMP-2 with rats demon-

strated that the HA/BMP hybrid showed

complete union bone repair and more

extensive bone formation compared with

HA alone or autografts.

3,7,25

Despite confirmation of the osteogenic

potential of BMP and the suitability of HA as

its carrier within animal trials, the clinical use

of BMP remains contentious. The aim of this

study was to investigate the optimal method

of loading BMP into an HA carrier. It has

been shown that BMP-2 remains stable at

temperatures up to 70

uC,

27

making plausible

the BMP incorporation during HA wet

precipitation synthesis. The present study

characterized and compared 3 methods of

loading BMP-2 into HA: (1) BMP incorpora-

tion during HA formation in a calcifying

solution, (2) immersion of HA powder in a

BMP solution, and (3) BMP incorporation

during

dicalcium

phosphate

dihydrate

(DCPD) conversion to HA. Comparison of

these methods was based on the extent to

which BMP could be loaded into the HA

carrier, the in vitro release profile of BMP

from the carrier, and the influence of BMP on

the physicochemical and morphologic prop-

erties of HA crystals in the carrier.

M

ATERIALS AND

M

ETHODS

Recombinant human bone morphogenetic

protein-2 produced by Escherichia coli was

purchased from GenScript (Piscataway, NJ) as a

sterile lyophilized powder that was reconsti-

tuted in 5 mM acetic acid. Sodium hydroxide,

acetic acid, calcium chloride, sodium phos-

phate monobasic monohydrate, and sodium

phosphate dibasic anhydrous were supplied

by MP Biomedicals (Solon, Ohio). Water used

throughout the project was purified by reverse

osmosis (Milli-Q, Millipore, Billerica, Mass).

Optimization of HA synthesis for BMP

incorporation into HA powder

A 10 mL solution of 2 M calcium chloride and

a 5 mL solution of 1.2 M sodium phosphate

dibasic anhydrous were prepared to obtain

a calcium (Ca)/phosphate (P) ratio of 1.67,

equal to that of stoichiometric HA. The

calcium chloride solution was slowly added

to the phosphate solution while stirring

using a magnetic stirrer. The pH of the

solution was adjusted to 8, and the solu-

tion was heated to one of the following

Rohanizadeh and Chung

Journal of Oral Implantology

661

temperatures: 37

uC, 40uC, 43uC, 45uC, 47uC,

and 50

uC. During heating, the solution was

covered to prevent evaporation. Stirring

continued for 2 hours to mature the

precipitated HA crystals. The mixture was
subsequently filtered quickly using a filter-

funnel and flask vacuum; this was followed

by rinsing once with 25 mL of warmed (50

uC)

water. The filter paper and the powder were

then removed from the funnel and were
placed immediately in an oven at 40

uC to be

dried overnight. The HA fabrication process

was repeated at a pH of 7.5 over the same

temperature range and conditions. The

synthesis condition that resulted in precipi-
tation of crystalline HA and that had the

lowest risk of causing BMP denaturation

(temperature and pH close to 37

uC and 7.4,

respectively, ie, physiologic conditions) was

chosen for BMP incorporation.

Preparation of DCPD and optimization

of DCPD-to-HA conversion for BMP

incorporation into HA powder

Two equimolar (0.8 M) 15 mL solutions of

calcium chloride and sodium phosphate diba-
sic anhydrous were prepared. Calcium chloride

was added to the phosphate solution at room

temperature, and the pH was adjusted to 5.

The solution was covered and was left to be

stirred at room temperature for 2 hours before
filtering and rinsing with 25 mL of room

temperature water. The DCPD powder was

dried overnight at room temperature.

Once dried, 1 g of the DCPD powder was

placed in 8.25 mL of 0.1 M sodium hydroxide
(dissolved as a buffered solution). The pH of

the solution was adjusted to 8, and hydrolytic

conversion was allowed to proceed at the

following temperatures: 37

uC, 40uC, 43uC,

45

uC, 47uC, and 50uC in an oven with shaker.

After 24 hours, the solution was removed,

filtered, and dried at 40

uC to retain the

powder. The entire process was repeated at

a pH of 8.5 and 9 at the same temperatures.

The reaction was also investigated at 50

uC

and at 60

uC at pH 9, 9.5, 10, and 11. A 0.2 M

solution of sodium hydroxide at 70

uC was also

trialed. Similarly, the DCPD-to-HA conversion

condition with the least risk of denaturing

BMP was selected for BMP incorporation.

Methods to load BMP into HA powder

N

Incorporation during HA precipitation: Based

on the results of optimization conditions for

HA synthesis (Table 1), 37

uC and pH 8 were

the selected parameters for this method.

BMP-2 solution was added to 5 mL unheat-

ed calcium chloride solution, which then

was slowly poured into the phosphate

solution (as described earlier) to obtain a

concentration of 120

m

g/mL of BMP in the

15 mL solution used for HA preparation. The

weight ratio between obtained HA and the

BMP available in the solution was approxi-

mately 1000:1; the same ratio was used

across all other BMP loading methods.

N

Immersion of HA powder in a BMP solution:

The second method of loading BMP into

HA involved immersing HA powder in a

solution containing BMP. A total of 700 mg

of preformed HA was immersed at 37

uC in

a 5.8 mL solution containing 120

m

g/mL

BMP-2, yielding the same HA/BMP weight

ratio and BMP concentration in solution as

T

ABLE

1

Product of calcium and phosphate precipitation at different pH values and temperatures as

determined by XRD*

pH

Temperature,

uC

37

40

43

45

47

50

7.5

DCPD

+ HA

DCPD

+ HA

HA

HA

HA

HA

8

HA

HA

HA

HA

HA

HA

*DCPD refers to dicalcium phosphate dehydrate; HA, hydroxyapatite.

Hydroxyapatite as a Carrier for BMP

662

Vol. XXXVII/No. Six/2011

the previous method. After 24 hours, the

solution was filtered and was rinsed with

25 mL warmed water (50

uC). The powder

was allowed to dry overnight at 37

uC.

N

Incorporation during DCPD-to-HA conver-

sion: None of the conditions tested to

convert DCPD to HA was successful (Table 2);

therefore based on previous works of Le-

Geros,

28

0.2 M sodium hydroxide solution at

70

uC was used for this method. A total of

120

m

g/mL of BMP was added to the 8.25 mL

of 0.2 M sodium hydroxide solution (70

uC).

The HA/BMP weight ratio and the BMP

concentration in solution were adjusted as

in the previously mentioned methods. The

conditions of DCPD-to-HA conversion were

described earlier.

BMP release profile

The amount of BMP incorporated into the HA

powder was determined by subtracting the

BMP concentration remaining in the calcifying

or immersion solution at the end of the

loading process (before filtration) from the

initial protein concentration of 120

m

g/mL. A

1.2 mM solution of sodium phosphate mono-

basic monohydrate adjusted to pH 7.4 was the

phosphate buffered solution (PBS) used for the

in vitro BMP release assay. The rate of BMP

release was determined by incubating 120 mg

BMP-loaded HA in 1.2 mL of PBS in an

Eppendorf tube at 37

uC in a shaker oven. In

all, 850

m

L of the supernatant was withdrawn

and frozen at 12 hours and at days 1, 3, 7, and

14. The removed supernatant was replaced

with 850

m

L of fresh PBS at each time point.

The experiment was set up for all 3 BMP-

loading methods using 5 samples per method

and time point. As a control, HA without BMP

was also incubated in PBS at 37

uC with

identical time points for supernatant with-

drawal. All collected supernatant was stored at

2

20

uC until the protein concentration was

measured using 2 different protein assays.

The Lavapep total protein fluorescence

assay (Fluorotechnics, Sydney, Australia) and

the Quantikine (R&D Systems Inc, Minneapo-

lis, Minn) BMP-2 enzyme-linked immunosor-

bent assay (ELISA) were used to determine

total protein uptake into HA and its release

profile in vitro. The microplate reader used for

both protein kits was the POLARstar OPTIMA

in conjunction with FLUOstar OPTIMA soft-

ware (BMG Labtech, Ortenberg, Germany).

Because calcium ions released from HA

powder may interfere with the measurement

of BMP concentration in the total protein

assay, 2 g of HA (without BMP) was soaked in

16.7 mL of water for 14 days. The supernatant

was removed and was used in making the

BMP stock solution and in the serial dilutions

to make standards for the total protein assay.

Physicochemical properties of HA (before

and after BMP loading)

X-ray diffraction (XRD) (Siemens D5000 X-ray

diffractometer, Siemens Healthcare Diagnostics,

Deerfield, Ill) was used to determine the crystal

T

ABLE

2

Product of hydrolytic conversion of DCPD at different pH values and temperatures as

determined by XRD*

pH

Temperature,

uC

37

40

43

45

47

50

60

8

DCPD

DCPD

DCPD

DCPD

DCPD

DCPD

–

8.5

DCPD

DCPD

DCPD

DCPD

DCPD

DCPD

–

9

–

–

–

–

–

DCPD

–

9.5

–

–

–

–

–

DCPD

–

10

–

–

–

–

–

DCPD

DCPD

11

–

–

–

–

–

DCPD

DCPD

*– refers to a condition not studied by clinical trial; DCPD, dicalcium phosphate dihydrate.

Rohanizadeh and Chung

Journal of Oral Implantology

663

structure of the obtained materials before and

after BMP loading. The parameters used were

40 kV and 30 mA, with a scan range between

10 and 40 degrees 2h and a step size of

0.02 degrees with 2 seconds per step. Scanning

electron microscopy (SEM) determined the

crystal morphology of the synthesized materials

and the effects of BMP incorporation on HA

crystal size and shape. The Zeiss Ultra Plus (Carl

Zeiss, Go¨ttingen, Germany) electron microscope

with a working distance of 4.4 mm and 20 kV

was used in this study.

R

ESULTS

Optimization of HA synthesis for

BMP loading

XRD analysis of powders obtained from the

calcium and phosphate precipitation reac-

tions demonstrated that a mixture of DCPD

and HA formed at pH 7.5 and at tempera-

tures of 37

uC and 40uC (Table 1). At temper-

atures above 43

uC, only HA crystals were

precipitated at pH 7.5. By increasing the pH

to 8, across all tested temperatures, the

materials obtained had only HA crystal

structure. Table 1 summarizes the products

obtained from precipitation in calcium and

phosphate solutions. For the second loading

method (DCPD-to-HA conversion), because

the conditions shown in Table 2 did not

elicit conversion of DCPD crystals to HA, the

DCPD powder was soaked in 0.2 M NaOH

(pH 13) at 70

uC to be hydrolyzed to HA.

28

Physicochemical properties of HA (before

and after BMP loading)

The typical XRD spectrum of HA precipitates

obtained from solution at pH 8 and 37

uC is

shown in Figure 1A. Diffraction peaks in this

spectrum are identified as those of HA. Major

peaks at 2h 5 26 degrees, 32 degrees, and

34 degrees indicate HA lattice planes of

(002), (211), and (300), respectively. Fig-

ure 1B and C shows the typical XRD spectra

of DCPD and HA converted from DCPD,

respectively. Figure 1B shows major peaks

at 2h 5 11.7 degrees, 21 degrees, and

F

IGURE

1. The X-ray diffraction (XRD) spectra of (A) hydroxyapatite (HA) precipitates at pH 8 and 37

uC; (B)

dicalcium phosphate dihydrate (DCPD) precipitates at pH 5 and room temperature; and (C) DCPD
converted to HA at pH 13 and 70

uC.

Hydroxyapatite as a Carrier for BMP

664

Vol. XXXVII/No. Six/2011

29 degrees, corresponding to DCPD lattice

planes of (020), (121), and (112) respectively.
Figure 1C identifies the typical HA peaks

after DCPD-to-HA conversion; no DCPD

peaks were observed after the conversion.

Following BMP incorporation via (1)

incorporation during HA precipitation, (2)

HA immersion in BMP solution, and (3)

incorporation during DCPD conversion to

HA, the XRD spectra (Figure 2A through C)
revealed that HA remained the only mineral

compound present, with major peaks at 2h

5

26 degrees and 32 degrees. The XRD

spectra showed a much higher level of noise

after BMP incorporation (Figure 2A through
C). No detectable alterations in the broad-

ness of these peaks of the XRD spectra were

observed following BMP loading.

SEM micrographs in Figure 3A through G

show crystal morphology of different groups

before and after BMP loading. The HA crystals

synthesized from wet precipitation were nano-

sized and mostly had a plate-like shape
(Figure 3A), whereas those formed from DCPD

conversion were more needle-shaped and

were larger, agglomerated, and more elongat-

ed (Figure 3C). The micrograph of DCPD

(Figure 3B) shows large (micron-sized) plate-

like crystals. The micrographs in Figure 3D

through G reveal morphologic changes in HA

crystals following BMP loading to HA powder.

The BMP incorporated during HA precipita-

tion reduced the size of the crystals but

retained the HA plate-like shape (Figure 3A vs

Figure 3D). After HA powder was immersed in

the BMP solution, in most areas, HA crystals

had a plate-like shape (Figure 3E); however, in

some areas, crystals appeared to be very small

and agglomerated compared with those

before BMP loading (Figure 3F: magnified of

boxed area in Figure 3E). BMP incorporated

during DCPD-to-HA conversion resulted in

little variation in the morphology of HA

crystals compared with those formed without

the presence of BMP (Figure 3C vs Figure 3G).

Release profile of BMP

The extent of BMP uptake into HA powder

was measured using ELISA and total protein

assays (summarized in Table 3). Based on the

F

IGURE

2. The X-ray diffraction (XRD) pattern of crystals after bone morphogenetic protein (BMP) uptake

via (A) incorporation during hydroxyapatite (HA) precipitation; (B) HA immersion in BMP solution; and
(C) incorporation during BMP dicalcium phosphate dihydrate (DCPD)-to-HA conversion.

Rohanizadeh and Chung

Journal of Oral Implantology

665

F

IGURE

3. Scanning electron micrograph (SEM) images of (A) hydroxyapatite (HA) precipitates at pH 8

and 37

uC; (B) dicalcium phosphate dihydrate (DCPD) precipitates at pH 5 and at room temperature; (C)

DCPD converted to HA at pH 13 and 70

uC; (D) HA crystals after bone morphogenetic protein (BMP)

incorporation during HA precipitation; (E) HA crystals after immersion in a BMP solution; (F) Figure 3E
boxed area magnified (HA crystals); and (G) HA crystals after BMP incorporation during DCPD-to-
HA conversion.

Hydroxyapatite as a Carrier for BMP

666

Vol. XXXVII/No. Six/2011

total protein assay, the highest percentage

of BMP uptake into HA was achieved with the

immersion technique followed by incorpora-

tion during HA precipitation, then DCPD-to-

HA conversion. When measured by total

protein assay, BMP uptake during the HA

immersion method was significantly higher

than that obtained using other loading

methods (Table 3). The ELISA assay demon-

strated significantly lower BMP uptake during

HA precipitation and comparable percentage

(around 96%) when BMP was loaded by

immersion and the DCPD-to-HA conversion

method. No significant differences were seen

when the amount of BMP in solution was

measured before and after the filtration

process, indicating that BMP adsorption on

filter materials and funnel was negligible.

Using the ELISA assay, the release profiles

of BMP were determined for all 3 BMP-

loading methods and are shown in Figure 4.

The total amount of BMP released after

14 days was 1028 6 181.6, 413.6 6 183.5,

and 619.3 6 139.8 ng/mL, respectively, for

HA precipitation, HA immersion, and DCPD-

to-HA conversion loading methods. The

percentage of BMP released (based on the

percentage of BMP uptake) after 14 days was

22.9 6 4.3, 0.43 6 0.18, and 1.23 6 0.28,

respectively, for each of the methods previ-

ously mentioned (Figure 5). All 3 BMP-

loading methods revealed a rapid increase

in BMP release during the first 12 hours

when compared with control. However, BMP

released for samples prepared using the

immersion method did not significantly

increase after 12 hours, unlike the 2 other

loading methods, which showed further BMP

release after 12 hours. Loading BMP into HA

powder during DCPD conversion showed a

secondary burst of BMP release at the 7 day

time point with minimal increase thereon,

whereas for samples in which BMP was

loaded during HA precipitation, the BMP

T

ABLE

3

Percentage of BMP uptake into HA measured by total protein and ELISA assays*

BMP Loading

Method

Incorporation During

HA Precipitation

HA Immersion in

BMP Solution

Incorporation During

DCPD Conversion to HA

Assay

Total Protein

ELISA

Total Protein

ELISA

Total Protein

ELISA

% BMP loaded

54 6 3.5

72 6 3.7

94.6 6 0.5

À

96.2 6 0.5

50.5 6 2.4

96.7 6 0.5

*BMP refers to bone morphogenetic protein; DCPD, dicalcium phosphate dehydrate; ELISA, enzyme-

linked immunosorbent assay; HA, hydroxyapatite.

À

Significantly higher than the other 2 BMP loading methods (n 5 5; ANOVA P , .001; post hoc

Tukey pairwise).

F

IGURE

4. Release profile of bone morphogenetic

protein (BMP) from hydroxyapatite (HA) powders
prepared using 3 BMP loading methods.

F

IGURE

5. Percentage of bone morphogenetic

protein (BMP) released from hydroxyapatite (HA)
powders, based on the extent of BMP uptake for
each respective BMP loading method.

Rohanizadeh and Chung

Journal of Oral Implantology

667

release profile continued to increase over the

14 day period.

D

ISCUSSION

Precipitation of calcium phosphates from

homogenous solutions requires the satura-

tion point to be reached, followed by

subsequent nucleation and crystal growth.

HA crystals are obtained in human physio-

logic conditions, alkaline conditions, and

temperatures above 37

uC; HA crystallinity is

increased by increasing the temperature in

the wet precipitation method.

29,30

Table 1

reflects these observations by showing that

increasing the acidity and decreasing the

temperature led to the precipitation of a

mixture of DCPD and HA. However, at a pH

of 8, only HA and no DCPD was formed. The

XRD pattern (Figure 1A) showed that the HA

obtained at a pH of 8 and at 37

uC was poorly

crystalline, demonstrated by broad peaks in

the diffraction spectra. The significance of

poorly crystalline HA is correlated with its

increased solubility, which may facilitate

BMP release once incorporated.

29

SEM micrograph (Figure 3A) showed that

HA crystals were nano-sized (500 3 100 nm)

plate shapes. The HA powder obtained had a

tendency to agglomerate, which has been

previously reported in wet precipitation of

HA.

31,32

Smaller HA crystals are desirable for

their expected increased bioactivity property

and their higher surface area for protein

adsorption.

32

In contrast to HA, DCPD pre-

cipitation from calcium and phosphate solu-

tions is predominant at a pH less than 6.5

and at room temperature.

33

Figure 1B shows

highly crystalline DCPD precipitates as seen

by sharp peaks in the XRD spectra when

formed under acidic conditions (pH 5) and at

room temperature. Figure 3B shows macro-

sized (up to 10

m

m 3 3

m

m) plate-like crystals

typical of DCPD crystal morphology. In

alkaline conditions, DCPD undergoes hydro-

lytic transformation to HA. The following

reaction details the hydrolysis of DCPD to

HA

34

:

10 CaHPO

4

.

2 H

2

O?

Ca

10

PO

4

ð

Þ

6

OH

ð

Þ

2

z18 H

2

Oz12 H

z

z4PO

3{
4

A range of temperatures (60

uC–140uC) and

pH values (6–14) have been investigated

to facilitate this hydrolysis reaction.

35

It was

seen that conditions at pH greater than 9

produced more elongated HA crystals. The

condition selected in this study—0.2 M

NaOH (pH 13) and 70

uC—are of an extreme

nature but were used here because the

threshold for DCPD transformation was not

determined (Table 2). At these conditions,

the HA produced showed different crystal

morphology with elongated rod particles (as

previously reported) but with reduced width

in comparison with HA crystals obtained

through direct wet precipitation (Figure 3C

vs Figure 3A).

BMP incorporation in HA has not been

specifically investigated in terms of the

physicochemical effects of BMP on HA.

However, the presence of other bone matrix

proteins, bovine serum albumin, and amy-

lase has retarded HA crystal growth when

present during precipitation of HA.

35–38

SEM

images in Figure 3D through G show smaller

HA crystals upon BMP uptake compared

with HA without BMP, supporting what has

been previously found with other proteins.

Incorporation of BMP during HA precipita-

tion (Figure 3D) reveals uniform rod needle-

like crystal morphology (approximately 200

3

10 nm) compared with HA precipitated in

the absence of BMP. The reduction in HA

crystal size in BMP loaded samples might be

due to binding of BMP to calcium sites in HA,

thereby inhibiting further crystal growth.

Samples prepared via immersion of HA

powder in BMP solution (Figure 3E) revealed

less uniform crystal morphology after BMP

uptake compared with those prepared by

other loading methods. In this group, most

crystals have a plate-like shape with crystals

Hydroxyapatite as a Carrier for BMP

668

Vol. XXXVII/No. Six/2011

of similar size to those obtained via BMP

incorporation during HA precipitation. Figure 3F

(magnified boxed area in Figure 3E) shows an

area of dissolution/reprecipitation of HA crystals

during the 24 hour immersion period in the

BMP solution. Dissolution of HA crystals results

in a local increase in calcium and phosphate

ions concentrations, leading to reprecipitation

of very small HA crystals (50 3 100 nm) off the

primary HA crystals.

38

These secondary crystals

showed different morphology and appeared

smaller and more agglomerated than the

primary HA crystals.

Unlike the other BMP-loading methods,

BMP incorporated into HA powders during

DCPD-to-HA conversion resulted in little

change in crystal morphology (Figure 3G).

The XRD spectra from all the methods in

the presence of BMP (Figure 2) show that

the XRD spectra with a much higher level of

noise compared with those prepared with-

out BMP (Figure 1) indicate the presence of

organic matter (such as BMP). The high level

of noise in XRD spectra made it difficult to

precisely determine the changes in peak

broadening; therefore, for all 3 groups, no

detectable changes in HA crystallinity were

observed after BMP loading.

BMP uptake

The amount of BMP uptake into HA powders

was determined via the protein assays seen

in Table 3. However, assumptions underlined

the calculated percentage of BMP uptake.

Foremost is the BMP concentration in

calcifying or immersion solutions, which

was taken to imply that the remaining BMP

was loaded into the HA powder. BMP

adsorption to filter paper, by sampling

before and after filtering, in each method

was considered and revealed little adsorp-

tion of BMP onto filter materials. However,

the washing step in every method may have

reduced the extent of the actual amount of

loaded BMP by removal of BMP, which was

loosely attached or incorporated. Finally,

BMP adsorption to the containers used for

BMP loading was not accounted for.

The amount of BMP uptake into HA via the

3 methods—incorporation during HA precipi-

tation, incorporation during DCPD-to-HA con-

version, and immersion of HA in BMP solu-

tion—varied significantly based on the method

of measuring protein in solution used (ELISA vs

total protein assays). The total protein assay

determines the state of BMP as total protein

(both denatured and undenatured protein

molecules), whereas the ELISA assay determines

the amount of BMP in the biologically active

conformation (undenatured molecules). Table 3

shows that the most extensive BMP loading

occurred during HA immersion in the BMP

solution, demonstrated by both ELISA and total

protein assays. This may have happened as a

result of the longer duration of BMP loading in

the immersion method (24 hours) compared

with the 2 hour interval for BMP incorporation

during HA precipitation. Moreover, HA crystals

immersed in the BMP solution experienced a

secondary HA crystal growth, allowing for BMP

adsorption onto the newly formed HA crystals,

increasing the overall amount of BMP loaded.

Based on the total protein assay, BMP uptake

during HA precipitation was the next highest

percentage. Unexpectedly, the DCPD-to-HA

conversion loading method resulted in the

lowest percentage, despite having a 24 hour

loading interval. HA formed via hydrolysis of

DCPD is known to be calcium deficient,

34

which may reduce the availability of BMP

binding sites to calcium, thereby reducing

the extent of BMP loading during DCPD-

to-HA. Moreover, the conversion reaction

involves DCPD dissolution and reprecipitation

of HA from calcium and phosphate ions.

However, precipitation of new HA crystals

does not take the allotted 24 hour period,

thereby reducing the time BMP had to bind to

calcium sites. More important, the HA precip-

itates obtained via DCPD conversion were

largest in comparison with those obtained by

the 2 other tested BMP loading methods.

Rohanizadeh and Chung

Journal of Oral Implantology

669

Therefore, the decrease in total surface area of

HA crystals for BMP adsorption may be

another factor in reduced BMP uptake in the

DCPD-to-HA conversion method.

It should be noted that the total protein

assay did not differentiate between dena-

tured and undenatured BMP molecules,

whereas the ELISA assay detected BMP only

in the biologically active conformation state.

The ELISA assay is unable to measure

denatured BMP molecules in the remaining

calcifying or immersion solution; therefore a

higher percentage of BMP was presumed to

be loaded into HA when the ELISA assay was

used, which is inaccurate. For this reason,

across all 3 BMP-loading methods, the

percentages of BMP uptake were elevated

when measured by ELISA assay rather than

the total protein assay (Table 3). Because the

total protein assay accounted for denatured

and active BMP, it gave a more accurate

depiction of BMP uptake into HA powder.

The ELISA assay showed that BMP uptake

for the DCPD-to-HA conversion method was

significantly greater than the respective value

obtained using the total protein assay. This

may be explained by the conditions used to

load BMP during DCPD-to-HA conversion. It

has been reported that after 8 hours of heat

treatment (70

uC), the activity of BMP was

significantly decreased.

27

Conversion of DCPD

powder to HA occurred at 70

uC over 24 hours,

increasing the percentage of denatured BMP

molecules and subsequently reducing their

detection in remaining solution via the ELISA

assay. This contrasts with BMP loading during

HA precipitation at 37

uC and pH 8, wherein

the secondary structure of BMP is better

maintained, allowing more accurate BMP

measurement in solution with ELISA and

thereby BMP uptake in HA powder.

BMP release profile

Unlike the ELISA assay, which is capable of

BMP-2 detection in the range of picogram,

the total protein assay used in this study was

unable to detect the very low concentration

of BMP released from HA powder. BMP

release from the samples prepared using

HA precipitation and DCPD conversion was

greater than in those prepared using HA

immersion in BMP solution. BMP uptake

during HA precipitation showed the best

release profile, with the most BMP (22.9%)

released in conjunction with a sustained and

prolonged release profile. The low release of

BMP (total of 0.43%) from samples prepared

by immersion in BMP solution could be due

to BMP detachment during washing caused

by loose adsorption of BMP on HA crystals.

BMP uptake during DCPD-to-HA conversion

showed a biphasic release profile with 2

stages of burst release, within the first

12 hours and 7 days. For this group, the

BMP release profile may indicate BMP initially

released from the HA crystals located on the

surface of HA agglomerates; after a week,

BMP was released from crystals located in

the inner regions of HA agglomerates.

However, the release profiles for all loading

methods showed an initial burst of BMP

release within the first 12 hours. It should be

noted that these release profiles reflect only

the amount of undenatured protein (detect-

ed by ELISA assay); the actual amount of

total released BMP (both denatured and

undenatured) is probably higher.

Based on results obtained in this study,

further optimization of BMP loading during

the HA precipitation method should be

considered.

Enhanced

understanding

of

BMP in HA adsorption would develop with

variation of the stage in which BMP can be

added during the HA precipitation process

(eg, in the calcium solution vs in the

phosphate solution vs during HA crystal

maturation). Upon optimization, the biolog-

ical osteogenic potential of these implants

would have to be evaluated through in vivo

trials. The addition of other bone augment-

ing drugs may be considered with HA as a

potential carrier.

Hydroxyapatite as a Carrier for BMP

670

Vol. XXXVII/No. Six/2011

Overall, these findings show the suitabil-

ity of HA as a BMP carrier. This study

highlights the necessity of investigating the

protein loading method because it directly

affects many properties of the carrier and its

ability to take up and release protein.

C

ONCLUSION

The potential for BMP in bone tissue

engineering remains limited so long as its

delivery is hampered. This study has dem-

onstrated different methods of combining

BMP, and that a single carrier, HA, can

produce great variation in protein uptake

and release, also affecting carrier physico-

chemical and morphologic properties. Based

on parameters for HA synthesis closest to

physiologic conditions, BMP incorporation

reduced HA crystal size during HA precipita-

tion (pH 8, 37

uC) and the immersion method.

However, protein loading during DCPD

conversion to HA (pH 13, 70

uC) showed no

significant alteration in HA crystals.

The negligible amount of BMP released

through the immersion method reflects the

need for future studies to optimize methods

of BMP loading, because this is the predom-

inant BMP loading method used in past and

current trials. BMP incorporation during HA

precipitation produced the best release

profile, despite not acquiring the highest

amount of BMP loaded. Its BMP release

profile could be described as slow, sustained,

and prolonged, as desired in BMP clinical

applications.

A

BBREVIATIONS

BMP: bone morphogenetic protein

DCPD: dicalcium phosphate dihydrate

HA: hydroxyapatite

ELISA: enzyme-linked immunosorbent assay

PBS: phosphate buffered solution

SEM: scanning electron microscopy

XRD: X-ray diffraction

A

CKNOWLEDGMENT

This work was supported by a research grant

from American Academy of Implant Dentistry

Research Foundation.

R

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