Hi,
Here is a good way to gain a primary Bone culture from rat Carnial tissue.
Not a complicate protocol.
I used Rat Calvaria from new born.
It works very good.

The protocol was taken from this paper:
Morphological and
Immunocytochemical
Characterization of Primary
Osteogenic Cell Cultures Derived
From Fetal Rat Cranial Tissue
From : Irie K et al. THE ANATOMICAL RECORD 252:554567 (1998)

Here is the protocol.
Cell Isolation and Primary Culture
of Osteogenic Cells
The experimental protocol and animal handling procedures
were approved by the Comite de deoutologie de
lexperimentation sur les animaux of Universite de Montreal.
Pregnant mothers were anesthetized with Metofane (methoxyfluorane;
Janssen Pharmaceutical, North York, ON, Canada),
and the fetuses were extracted by cesarean section and
sacrificed by decapitation. Newborn rats were similarly anesthetized
and sacrificed. Cells were isolated by sequential
enzymatic digestion from 1516-day fetal and 2-day newborn
cranial tissue ofWistar rats (Charles River, St. Constant, QC,
Canada). Briefly, for fetal cell cultures, the skin and underlying
connective tissue were stripped off the developing brain
and mechanically separated from each other with forceps. For
newborn calvariae, the periosteum was removed and the bone
tissue distant from the sutures was cut into small pieces with
scissors. Both the fetal cranial connective tissue and the pieces
of postnatal calvarial bone were then digested with a mixture
of enzymes consisting of 0.2% collagenase (Boehringer Mannheim,
Laval, QC, Canada) and 0.25% trypsin (Gibco, Burlington,
ON, Canada) at 37°C for sequential periods of 5, 15, and
25 min. In each case, the cells from digests 2 and 3 were pooled
and filtered with a 200?Mmetal mesh screen and plated in 35
mm multi-well Falcon polystyrene dishes (Fisher Scientific,
Montreal, QC, Canada) or on Thermanox coverslips (Fisher)
at a density of 60,000 cells/well. Some cells were also plated in
silicone dishes of similar size, prepared using the Sylgard
184 silicone kit (Dow Corning Canada Inc., Mississauga,
ON, Canada) and treated with 1N HCl for 30 min.
Initially, the cells were grown in MEM with Earls salts
(Gibco) supplemented with 10% fetal calf serum (Gibco)
and penicillin (100 U/ml)/streptomycin (100 ?g/ml; Gibco)
at 37°C in a humidified atmosphere with 5% CO2. Twentyfive
?g/ml of ascorbic acid (Sigma Chemical Co., St. Louis,
MO) was added on day 3. On day 7, the ascorbic acid
concentration was raised to 50 ?g/ml and 10mMb-glycerophosphate
(Sigma) was also added. The medium was then
changed every 3 days. The progression of the cultures was
followed using an Olympus IMT-2 inverted light microscope.
At 7, 14, and 21 days, some cultures were processed
for ultrastructural and (immuno)cytochemical analyses.