Alternated activation with relaxation of periosteum stimulates bone modeling and remodeling

Gradual elevation of the periosteum from the original bone surface, based on the principle of distraction osteogenesis, induces endogenous hard and soft tissue formation. This study aimed to assess the impact of alternating protocols of activation with relaxation (periosteal pumping) on bone modeling and remodeling. One hundred and sixty-two adult male Wistar rats were used in this study. Four test groups with different pumping protocols were created based on the relaxation applied. Two control groups underwent an activation period without relaxation or only a single activation. One group was sham-operated. Periosteal pumping without period of activation induced gene expression in bone and bone remodeling, and following activation period enhanced bone modeling. Four test groups and control group with activation period equaled the values of bone modeling at the end-consolidation period, showing significant downregulation of Sost in the bone and periosteum compared to that in the sham group (p < 0.001 and p < 0.001, respectively). When all test groups were pooled together, plate elevation from the bony surface increased bone remodeling on day 45 of the observation period (p = 0.003). Furthermore, bone modeling was significantly affected by plate elevation on days 17 and 45 (p = 0.047 and p = 0.005, respectively) and by pumping protocol on day 31 (p = 0.042). Periosteal pumping was beneficial for increasing bone repair when the periosteum remained in contact with the underlaying bony surface during the manipulation period. Following periosteal elevation, periosteal pumping accelerated bone formation from the bony surface by the modeling process.


Qualitative histological analysis
Signs of locally confined inflammatory reaction were observed in five animals: three animals (two LD and one sham group) on day 4 and two animals (LD group) on day 7 of the observation period.Histological analysis revealed signs of minor infection next to the distraction screw in nine animals: five animals (two PDO, one D_PP, one PP, and one PE_1) on day 17, one animal (PDO) on day 31, and three animals (DDP, PP, and PE_1) on day 45 of the observation period.Minute exposure of the device was observed in one animal (PP_D) on day 31 and one animal (PDO) on day 45 of the observation period.All the samples were included in the analysis.
Bone formation to different extents throughout the observation period was observed in all animals.The gap between the new bone and the distraction plate was occupied by loose connective tissue.The type of new bone was woven bone at the leading edge of the bone apposition.Signs of bone maturation were observed over time in all groups.The maximal values of newly formed bone for all groups with distraction plates were found in region of interest_2 (ROI_2, the higher part of the distraction gap), with comparable values observed in ROI_1 and ROI_3.The original calvarial bone mainly comprised compact bone with intervening marrow cavities.Bone marrow was present in the middle part of the calvaria and was generally more extended in ROI_2.The old calvarial bone and newly formed bone were occasionally connected to the bone marrow cavities and blood vessels, especially in ROI_2.When the calvarial bone was thin, the bone marrow was presented as small isolated islands or was completely absent, as sporadically observed in ROI_1.Differences were observed in bone modeling and remodeling between the groups.

Latency period
The CNNB1 in the LD group was significantly overexpressed in the bone (+ 53% vs. sham) and periosteum (+ 65% vs. sham) on day 1 of the observation period (Table 1).Sost expression in the bone was also upregulated in the LD group on day 1 of the observation period (+ 429% vs. sham), reaching the significant effect for the device placement, healing period, and their interactions.The bone expression of CNNB1, Runx2, Sparc, ACP5, and BMP2 on day 4 of the observation period in the LD group was downregulated (− 62%, − 36%, − 36%, − 59%, and − 81%, respectively vs. sham).Periosteal expression of the ACP5 in the LD group was significantly upregulated, while Sost in the periosteum was not expressed (Table 1).
The swelling was more intense in the LD group than in the sham group on days 1 and 4 of the observation period and declined on day 7 (Fig. 1).A distinct periosteal layer was discernible in both groups and was sporadically disrupted in the LD group.Intensive coagulum formation was generally observed above the periosteum during the latency period, independent of device placement (Supplemental Fig. S1).Signs of minimal bone formation between the periosteum and the original bone surface were observed on day 4 of the observation period in both groups (Supplemental Fig. S2).The MBBF was consistently higher in the sham group than in the LD group, reaching significance for the newly formed woven bone (WB), bone marrow (BM), and total new bone area (TNB) on day 4 of the observation period (Table 2).Device placement, healing period, and their interaction negatively affected the MBBF.On day 7 of the observation period, patch-wise deposition of primary woven bone covered with osteoid and osteoblasts was observed in both groups (Supplemental Fig. S2).Osteoclasts were occasionally observed in the LD group at the sites where the device contacted the original bone surface.Minimal signs of bone turnover on days 4 and 7 of the observation periods were always associated with the presence of BM (Fig. 1, Supplemental Fig. S1).Device placement negatively affected relative calvarial bone marrow (R_CBM) on day 4 of the observation period.
The micro-computed tomography (μCT) parameters varied between the groups.Cortical porosity (Ct.Po) and marrow volume (Ma.V) were lower in the LD group than in the sham group; however, none of the parameters showed a statistically significant difference.
The newly formed bone comprised woven bone with bone cavities (Fig. 2).A contiguous layer of new bone was sporadically reinforced by the parallel-fibered bone (Fig. 3).Signs of calvarial bone resorption and apposition were observed at the sites where the tip of the distraction screw touched the calvarial bone surface (Supplemental Fig. S3).Orbicular bone structures facing soft tissues with many osteoblasts and osteoids indicate ongoing bone formation.The greatest MBBF was observed in the PPD, PP_D, and D_PP groups (Fig. 2), with the D_PP group showing a highly significant difference for NB, BM, and TNB compared to the sham group (Fig. 4).The groups with plate elevation (W/PE) demonstrated higher WB (p ≤ 0.05), TNB (p = 0.047), and relative calvarial new bone (R_CNB, p = 0.04) values than the groups without plate elevation (Wo/PE), whereas no significant effect was    4e).More intensive bone remodeling was observed in all groups compared to the latency period, especially in ROI_2.
The W/PE group demonstrated higher cortical thickness (Ct.Th, p = 0.017) and cortical tissue volume (Ct.TV, p = 0.049) than the Wo/PE group.The PP_D and PE_1 groups had the highest Ct.Th, cortical bone volume (Ct.BV), and Ct.TV values (Fig. 4).Furthermore, an inverse relationship was detected in the PE_1 and D_PP groups for tissue mineral density (TMD) and Ct.Po values, with the lowest and highest values, respectively.The pumping protocol did not significantly affect the µCT parameters.
The newly formed woven bone in all the groups contained bone cavities with immature bone marrow reinforced with parallel-fiber bone (Fig. 5).A gradient in bone maturation was evident, with more mature bone moving towards the old calvarial bone.Osteoid-lined bone marrow cavities were present along the woven bone surface.Bone resorption sites were recognizable at contact sites between the distraction rod and bone surface, with intensive bone formation (Fig. 5, Supplemental Fig. S3).Fine orbicular structures towards the skin indicated intensive bone formation (Fig. 3).The W/PE group had more BM (p = 0.020) than the Wo/PE group, whereas the groups with pumping protocol (W/PP) had more BM (p = 0.024), new lamellar bone (LB, p = 0.008), and TNB (p = 0.042) than the groups without pumping protocol (Wo/PP).The D_PP group demonstrated the most intense signs of bone formation (Fig. 5), with the highest WB, LB, BM, TNB, and R_CNB values and the lowest relative calvarial old bone R_COB value (Fig. 6).Table 3. Gene expression in bone during consolidation period.Means ± SD are shown.PDO = 1 activation every 24 h for 7 days; PDP = 2 activations/1 relaxation every 12 h for 10 days; D_PP = 1 activation every 24 h for 7 days followed by 1 activation/1 relaxation every 12 h for 3 days; PDP = 1 activation/1 relaxation every 12 h for 3 days followed by 1 activation every 24 h for 7 days; PP = 1 activation/1 relaxation every 12 h for 10 days; PE_1 = 1 activation; Sham = flap elevation without device placement.17 days = end-activation period; 31 days = mid-consolidation period; 45 days = late-consolidation period.Significant values are in [bold].www.nature.com/scientificreports/Plate elevation showed no significant effect on the µCT parameters.Except for TMD, all µCT parameters were significantly higher in the W/PP group than in the Wo/PP group, reaching significance for Ct.Th (p = 0.030), Ct.BV (p = 0.018), and Ct.TV (p = 0.020).Furthermore, the PP group had higher Cr.Th, Ct.BV, and Ct.TV values than the PE_1 group and Cr.Th and Ma.V values than the sham group (Fig. 6).

Day 45 of the observation period (end-consolidation)
The Coll1a bone expression was highest in the PDO group (+ 34% vs. sham).Nevertheless, bone genes were generally downregulated compared to those in the sham group (Table 3).Furthermore, Sost was highly expressed in the sham group.CNNB1 and BMP2 periosteum expression was downregulated in the test groups, being lowest in the D_PP and DDP groups (− 91% and − 92%, respectively, vs. sham; Table 4).Sost expression was significantly upregulated in the sham group.
Woven bone formation with osteoids and numerous osteoblasts was observed beneath the distraction plate (Figs. 3 and 7).Bone resorption with Howship's lacunae and osteoclasts was still observed in the distraction screw region, with bone formation occurring next to the resorption sites (Fig. 7, Supplemental Fig. S3).The new bone layers in the three control groups were more mature.The new bone outside the plate consisted mainly of lamellar bone, cavities with mature bone marrow, and even surface contours without major signs of resorption (Fig. 3).The extent of bone formation among the groups with plate elevation was similar, reaching statistical significance for TNB, WB, and LB compared to the sham group (Fig. 8).The W/PE group showed significantly higher LB (P = 0.004), BM (p = 0.032), TNB (p = 0.005), and R_CNB (p = 0.003) values than the Wo/PE group, whereas no significant effect was detected for the pumping protocol.Sham group showed higher R_COB and lower R_CNB values than those in the PP group (Fig. 8).The highest R_CNB values in the PP group were also detected in the ROI_MS (Supplemental Fig. S4).
No significant effect was detected for plate elevation or pumping protocol on the µCT parameters.The PDO group showed the highest Ct.Th, Ct.BV, and Ct.TV values, which significantly differed for the Ct.TV in the PE_1 and sham groups (Fig. 8).Table 4. Gene expression in periosteum during consolidation period.Means ± SD are shown.PDO = 1 activation every 24 h for 7 days; PDP = 2 activations/1 relaxation every 12 h for 10 days; D_PP = 1 activation every 24 h for 7 days followed by 1 activation/1 relaxation every 12 h for 3 days; PDP = 1 activation/1 relaxation every 12 h for 3 days followed by 1 activation every 24 h for 7 days; PP = 1 activation/1 relaxation every 12 h for 10 days; PE_1 = 1 activation; Sham = flap elevation without device placement.17 days = end-activation period; 31 days = mid-consolidation period; 45 days = late-consolidation period.Significant values are in [bold].

Discussion
The present study's results indicate that the pumping protocol before the gradual elevation of the periosteum enhanced the MBBF at the mid-consolidation period, and without periosteal elevation the RBBF at the endconsolidation period.This, in turn, means that the hypothesis of the present study was confirmed.The space provided by the distraction plate positively affected the MBBF, as all groups with elevated plate had similar amounts of newly formed bone at the end-consolidation period.The model used in the present study is complex, as the process of periosteal wound healing is conditioned by mechanical stimuli.
Placing the distraction device during surgery disturbed the local environment.Device placement, healing period, and interaction significantly affected all three histomorphometric parameters.The upregulation of CNNB1 in the LD group one day after surgery suggests the activation of the Wnt/β-catenin signaling pathway, essential in regional anabolic response to loading 29 .Device placement, healing period, and interaction significantly affected the expression of Sost in bone, a potent inhibitor of the Wnt/β-catenin pathway.Sost expression is specific to terminally differentiated cells embedded within a mineralized matrix 30,31 .Sost binds to the first β-propeller in the extracellular domain of LRP5 and LRP6 and interferes with the formation of the LRP/Wnt/ FZD heterotrimer.The expression of CNNB1 in the bone was significantly downregulated in the LD group on day 4 of the observation period.Furthermore, device placement negatively affected the expression of other osteogenic genes and all MBBF parameters on day 4 of the observation period.Sost has been identified as a key regulator of bone formation that inhibits osteoblast differentiation 32 and stimulates osteoclast differentiation in a RANKL-dependent manner 33 .Sost expression in the periosteum was not detected in any of the samples.This was expected, as Sost is an osteocyte-specific protein 34 .
At the end of the activation period, all groups with device placement showed an increased MBBF compared to the sham group.MBBF depends mainly on the ability of the lining cells to form new bone upon stimulation with mechanical forces.Manipulation of the distraction plate significantly upregulated the periosteal expression of CNNB1, RunX2, and Sparc in the periosteum and Coll1a expression in the bone and periosteum, reaching the highest levels in the PP group.Maintaining contact between the periosteum and the underlying bone seems to play an important role in the upregulation of genes in the periosteum.However, the expression of RunX2 was upregulated in the PE_1 group.Mechanical stimuli are key determinants of bone mass; even low-magnitude mechanical stimulation has an anabolic effect on bones 35 .The effected movement and loading of the distraction plate was not controlled, as the manipulation of the distraction rod was performed manually.The instability of the distraction plate may also explain the higher MBBF values in the PE_1 group than in the sham group.In contrast, the sham group showed more R_CNB than the PE_1 group.Furthermore, the sham group showed  the highest bone marrow area within the calvaria.Histologically observed signs of bone turnover were always related to the presence of the bone marrow.Decreased bone marrow may indicate adaptation of skeletal tissues to changes in the mechanical environment 36 .However, MBBF activation may be accompanied by RBBF prevention.This seemingly paradoxical observation in groups with plate elevation may be explained by the balance of bone formation and resorption at the periosteal and endosteal surfaces 37,38 .Alterations in cortical envelopes upon loading can accelerate the modeling of periosteal bone formation and endocortical resorption drift 39,40 .However, the highest Ct.Po in the D_PP group may be related to increased periosteal apposition and intracortical and endocortical remodeling 41 .
The PP group showed the highest expression of all evaluated genes in the bone at day 31 of the observation period.Thus, the importance of intimate contact between the periosteum and the underlying bone is confirmed for gene expression in the bone.The protocol in the PP group may have initiated Frost's regional acceleratory phenomenon that enhances all ongoing regional processes, modeling and remodeling, and healing of hard and soft tissues 42 .Following the initial activation, there is a subsequent propagation of the "load signal" to adjacent osteocytes, connected to the decreased expression of the Wnt ligand inhibitors in the cells that subsequently activate β-catenin signaling 29 .Deeply embedded osteocytes in the mid-cortical region are the first to be activated  by a biochemical load signal generated from the bone surface 29 .The expression of CNNB1, Postn, and Sost in the bone was upregulated in the PP group without reaching significance for Sost; possible reasons for this may be the limited number of samples per group and the healing period.Nonetheless, Postn has a dual capacity to inhibit Sost expression and directly induce Wnt-b-catenin activation 43 .Furthermore, Postn mediates local MBBF and RBBF processes in response to fatigue loading 44 .As bone resorption decreased and bone formation relatively increased at day 31, the positive balance of local bone turnover increased RBBF 39 .The D_PP group, but not the PP group, exhibited a dual effect on both RBBF and MBBF.A possible explanation for this discrepancy in the lowest gene expression may be the time necessary for the progression of bone turnover as observed histologically.MBBF can be estimated as a part of the extensive RBBF, where a new bone matrix is formed over the peripheral area of the previous resorption lacunae, such as spilling over 45 .On day 45 of the observation period, all groups with plate elevation levelled the TNB values of the D_PP achieved on day 31.The same plate elevation distance in these groups confirms the importance of space maintenance over a longer healing period 46 .Bone expression of Sost was downregulated in all groups compared to that in the sham group.Osteocytes respond to mechanical loading by decreasing sclerostin production 36,47 , which is increased by unloading 47 .An elevated level of Sost contributes to the absence of callus formation 30 days after  fatigue 44 .Furthermore, Sost was expressed in the periosteum of the sham group.Other than osteocytes, several cell types can express Sost 34 .The minimal RBBF and MBBF values confirm that the sham group reached a steady state of bone homeostasis 48 .The highest RBBF was observed in the PP group two weeks after gene expression was induced, as previously observed in the D_PP group.The delay in RBBF compared to MBBF may be explained by the cortical bone of the calvaria with a small amount of bone marrow (without bone trabeculae), which differs in response to mechanical loading compared to the trabecular bone 39 .The highly significant difference achieved in the PP group suggests that considering specific indications, PP may be successfully performed to enhance both RBBF and MBBF, with the plates maintaining contact with the original bone.The lowest Ct.Po in the D_PP group indicated that fused lacunae were eventually replaced by a larger volume of a newly formed matrix that underwent primary and slower secondary mineralization 49 .Periosteal woven bone formation aids in rapid recovery from 15 to 30 days after fatigue loading 44 .The bone cells in the pumping groups on day 45 of the observation period may still be in the transient stage, as indicated by the presence of woven bone on the leading front of bone apposition 46 .Longer observation periods may be required to complete the remodeling process through the feedback loop of the mechanostat.A simple and effective treatment approach is required for patients with extended bone deficiency.Endogenous stimulation of the MBBF and RBBF by the pumping protocol suggests a possible therapeutic role for the mechanically stimulated periosteum, avoiding the need for exogenous growth factors or stem cells.However, the wound dehiscence in the periosteal distraction remain the risk for unfavorable wound healing.Signs of exposure of the distraction plate were observed in the present study; nevertheless, the distraction seemed to be advantageous for the absence of major infection.Maximizing the activity of intrinsic growth factors may have significant clinical relevance as an alternative to more complex bone regeneration procedures.Prior to clinical application, several limitations should be still addressed.Detection of relevant proteins within the distraction gap utilizing immunohistochemistry or in situ hybridization will be necessary to confirm the RT-PCR results and address the knowledge gaps across the repair window.Analysis of an alternative mechanism involving crosstalk with other signaling pathways is warranted for a more comprehensive understanding of the repair processes.Since age imposes a significant variation on bone properties, additional studies are required beyond the young adult stage.

Conclusions
The findings from this complex model of bone healing confirm the fundamental rule that mechanical stimulation can govern endogenous bone formation.Elevation of the periosteum from the bone surface significantly affected the MBBF and RBBF on days 17 and 45 of the observation period and the inclusion of the pumping protocol on day 31.Furthermore, the impact of PP on the osteogenic process depended on the relationship between the periosteum and the underlying bone surface.Maintenance of contact between the periosteum and the bone upregulated gene expression and RBBF, whereas periosteal elevation from the bony surface significantly enhanced MBBF.The link between Wnt pathway activation and mechanically induced RBBF and MBBF was confirmed, at least in part, via the downregulation of Sost.

Animals
All methods were carried out in accordance with the guidelines of the Institutional Animal Care Committee and reported in compliance with the ARRIVE guidelines.The study protocol was approved by the Committee for Animal Research, State of Bern, Switzerland (approval no.31/18).One hundred and sixty-two 14-week-old male Wistar rats were acclimated for 14 days, housed in a room with an adjusted climate (temperature, 22-24 °C ± 2 °C; humidity, 30-60% ± 5%) and special sun-substitution ultraviolet light (12 h photoperiod, 6 h-18 h) without excessive or surprising noise.Three rats were housed in individually ventillated cages and fed a standard rodent diet and water ad libitum.

Study design
Two groups of animals (N = 36, n = 18/group) were observed during latency period, with device placement (LD group) and without device placement (sham group).The animals of both groups were euthanized after one (n = 6/ group), four (n = 6/group), and seven days (n = 6/group) following surgery.Seven groups of animals (N = 126, n = 18/group) were subjected to the different treatment modalities, and euthanized 17 (end-activation, n = 6/ group), 31 (mid-consolidation, n = 6/group), and 45 days (end-consolidation period, n = 6/group) after surgery.Animals were randomized using a software package with a systematic randomization protocol (www.rando mizat ion.com).Potential confounders were not specifically controlled.
Sample size was calculated with an a-priori power model a level of significance a = 0.05 and error ß = 0.20 (power 1 − ß = 0.80), a peri-operatory risk of about 5% and a generic inter-individual variability.According to the power calculation, total sample size of 3 animals per subgroup were required to be 80% sure that the limits of a two-side 95% confidence interval will exclude a difference between the treatment modalities of more than 5%.

Anesthesia and surgery
Anesthesia and surgical procedures were performed as previously described 27 .Anesthesia was induced with 8% isoflurane (Attane™, MINRAD Inc., Orchard Park, NY, USA) and 600 ml/min oxygen in the induction chamber.The rats were placed in the prone position with a non-rebreather facemask (half-open system), maintaining a flow of 3-6% isoflurane with 200 ml/min oxygen.Using an aseptic technique (shaving of the operative area and disinfection with Betadine), local anesthesia with 10% Xylocaine Spray (AstraZeneca, Zug, Switzerland) was administered in the operative field.A mid-sagittal incision was made through the skin and periosteum, and the flaps were carefully reflected from the forehead to expose the calvarial bone (Fig. 9).The distraction devices were placed on the calvarial bones.The periosteum and the skin were sutured over the distraction device in two layers.No device was placed in the sham-operated group.The depth of anaesthesia was determined by observing vital parameters and movement after pain stimulation.A peri-operative dose of 0.1 mg/kg buprenorphin s.c.(Tamgesic®, ESSEX Chemie, Luzern, Switzerland) was continued for four days post-operatively.The animals were monitored daily during the first three weeks and every second day after that until euthanasia on health symptomatology using standardized score sheets.
The distraction device was manipulated under inhalation anesthesia (6% isoflurane and 600 ml/min oxygen).The distraction screw was activated manually by using a hex key.Turning the distraction screw for a quarter of a complete turn (90°) resulted in a 0.1 mm elevation of the plate 27 .For relaxation, the distraction screw was turned back to the same amount as that used for activation.PP protocol consisted of alternated activation with relaxation every 12 h, and the distraction protocol of activation only every 24 h.Animals were subjected to one of the following protocols: • PDO: distraction (activation every 24 h) during seven days • DDP group: distraction (activation after 12 h and 24 h) alternated with relaxation (after 36 h) for ten days • PP_D: PP (alteration of activation and relaxation every 12 h) for three days, followed by distraction (activa- tion at 24 h) for seven days • D_PP: distraction (activation once in 24 h) for seven days, followed by PP (alteration of activation and relaxa- tion at 12 h) for three days • PP: PP (alteration of activation and relaxation every 12 h) for ten days • PE_1: single activation • Sham: sham-operated animals without device placement The detailed protocol of device manipulation is presented in Supplementary Table 1.

Euthanasia
Six rats from each group were euthanized at six observation periods (1, 4, 7, 17, 31, and 45 days after surgery).The animals were euthanized by an overdose of gaseous carbon dioxide in an empty Plexiglas box and observed for at least 10 min after cardiac arrest and breathing cessation.Three animals from each group were used for the real time quantitative polymerase chain reaction (RT-qPCR) analysis.The recovered samples of the other three animals were excised and immediately immersed in a 10% buffered formaldehyde solution with 1% CaCl 2 .The specimens were processed for the µCT and histological analysis.

RT-qPCR
The skin was elevated from the head, and two samples were collected from each site: the periosteum and bone.The periosteum was incised at the base of the distraction gap and was carefully removed from the distraction device (first sample).The device was then removed.The bone fragment corresponding to the gap region was osteotomized using an oscillating saw and excised from the calvaria.Full bone thickness was used for the analysis (second sample).

µCT analysis
The distraction sites were subjected to radiography (25 kVP for 10 s) in two projections using a desktop conebeam scanner (µCT 40, Scanco Medical AG, Brüttisellen, Switzerland).The X-ray source (E) set at 70 kVp with 114 mA at high resolution (1000 projections/180º) showed an image matrix of 2048 × 2048 pixels.The diameter of the sample holder that allowed an increment (resolution) of 15 µm (= voxel size) was 30.7 mm.The integration time was set as 3 s.The µCT slices (1000) were reconstructed perpendicularly to the sagittal axis of the calvarium.Mineralized tissue was selected from grayscale images (0-1000) with a specific threshold of 220 per milles, corresponding to a value of 530 mg HA/cm 3 .Voxels above this value were categorized as mineralized bone, background, or titanium.The entire bone segment underneath the distraction plate was included in the analysis comprising pristine, calvarial bone and newly formed bone, as it was impossible to distinguish old from new bone at the later stages of healing.VOI_Summ was measured as the sum of the three volumes of interest (VOIs) outlined in the lower part of the distraction gap (VOI_1, 0-5 mm), the higher part of the distraction gap (VOI_2, 5-10 mm), and outside the distraction plate (VOI_3, 10-15 mm; Fig. 10a).The reconstructed 2D images were evaluated by 3D segmentation of the VOI, Gaussian Sigma at 0.8, and Gauss support at 1. Cortical contouring was automatically defined using Amira software (Amira, Visualization Sciences Group, Düsseldorf, Germany).For cortical analysis, the following structural parameters were generated: Ct.Th (μm), Ct.BV (mm 3 ), Ct.TV (mm 3 ), TMD (mg HA/cm 3 ), and Ct.Po (%).Ma.V (mm 3 ) was calculated as follows: Ct.TV-Ct.BV.

Histological analysis
The samples were rinsed in running tap water, dehydrated in ascending concentrations of ethanol (40-100%) and xylol and embedded in methylmethacrylate.The embedded tissue blocks were cut along the axis of the distraction device into approximately 800 μm-thick sections using a slow-speed diamond saw (Varicut® VC-50, LECO, St. were measured separately and selected manually, using four region of interests (ROIs).The ROI_Sum was measured as a sum of the lower part of the distraction gap (ROI_1, 0-5 mm), higher part of the distraction gap (ROI_2, 5-10 mm) and outside the distraction plate (ROI_3).In addition, a ROI corresponding to of the micro-screw region (ROI_MS) was measured distally to the ROI_1.(C) The histological sections outside the distraction plate were projected parallel to the central sections, and the ROIs outlined using the same reference lines.

Figure 1 .
Figure 1.Representative transversal histological images of the calvarium illustrating sections in the midaxis (above) and outside the distraction plate (below) in the DC and Sham groups at 1-day, 4-day and 7-day observation periods (latency period).The boxed areas in overviews (left) are magnified (right), showing calvarial bone (CB) with bone marrow (BM) and blood vessels (BV).Signs of bone remodeling (arrowheads) are observed lining the bone marrow cavities at 4-day and 7-day observation periods.Remnants of blood clot (C) are visible at all three observation periods.Toluidine blue and fuchsin staining.

Figure 2 .
Figure 2. Representative transversal histological images of the calvarium illustrating sections in the midaxis (left) and outside the distraction plate (right) at 17-day observation period (end-distraction).The overviews demonstrating minor new bone apposition (arrowheads) on top of the original, calvarial bone (CB).Remnants of blood clot (C) are sporadically observed.The boxed areas in overviews are magnified, showing signs of bone remodeling (arrowheads) adjacent to the bone marrow (BM).Blood vessels (BV) are connecting calvarial bone with newly formed woven bone (NB).Toluidine blue and fuchsin staining.

Figure 3 .
Figure 3. Representative transversal histological images of the calvarium illustrating new bone (NB) apposition on top of the original, calvarial bone (CB) in ROI_3 outside the distraction plate at 17-day, 31-day and 45-day observation periods.Newly formed bone is covered with the periosteum (arrowheads).The boxed areas (left) are magnified (right).New bone at 17-day observation period is mainly composted of the woven bone covered with the osteoid (arrows).At the 31-day observation period, newly formed bone is reinforced by parallelfibred bone (*) and contain bone cavities with immature bone marrow (BM) and blood vessels (BV).New dense, lamellar bone (*) was observed in all groups at the 45-day observation period.A clear gradient in bone maturation was evident especially in the D_PP group, with woven bone (WB) indicative of ongoing bone formation.

Figure 5 .
Figure 5. Representative transversal histological images of the calvarium illustrating sections in the midaxis (left) and outside the distraction plate (right) at 31-day observation period (mid-consolidation).The overviews showing bone formation (arrowheads) on the original, calvarial bone (CB).The boxed areas in overviews are magnified, demonstrating bone marrow (BM) and blood vessels (BV).Signs of bone remodeling (arrowheads) are seen next to the bone marrow.Bone resorbtion is observed at sites where distraction rod was in contact with the calvarial bone (arrows).Occasionally, bone marrow is intervening between the calvarial bone and newly formed bone (NB).Toluidine blue and fuchsin staining.

Figure 7 .
Figure 7. Representative transversal histological images of the calvarium illustrating sections in the midaxis (left) and outside the distraction plate (right) at 45-day observation period (end-consolidation).The overviews demonstrating newly formed bone (arrowheads) on top of the original, calvarial bone (CB).The boxed areas in overviews are magnified, showing signs of bone remodeling (arrowheads) adjacent to the bone marrow (BM) and blood vessels (BV).Occasionally, blood vessels are connecting calvarial bone with newly formed bone (NB).Resorbtion of the calvarial bone (arrows) is seen at sites in contact with the distraction rod.Toluidine blue and fuchsin staining.

Figure 9 .
Figure 9. Intraoperative view of the surgical area following flap elevation (A).The periosteum was elevated (B) and the distraction device with perforated plate placed (C).The periosteum (D) and the skin (E) are closed with interruptive sutures in two layers.Clinical appearance of the treated sites at 17-day (F), 31-day (G) and 45-day (H) observation periods.

Figure 10 .
Figure 10.Micro-CT and histomorphometric analysis.(A) The volume of interest (VOI) on the micro-CT was measured as a sum of three VOIs: lower part of the distraction gap (VOI_1, 0-5 mm), higher part of the distraction gap (VOI_2, 5-10 mm) and outside the distraction plate (VOI_3).(B) The area parameters for the histomorphometric analysis on the central sections for bone modeling (yellow) and bone remodeling (blue)were measured separately and selected manually, using four region of interests (ROIs).The ROI_Sum was measured as a sum of the lower part of the distraction gap (ROI_1, 0-5 mm), higher part of the distraction gap (ROI_2, 5-10 mm) and outside the distraction plate (ROI_3).In addition, a ROI corresponding to of the micro-screw region (ROI_MS) was measured distally to the ROI_1.(C) The histological sections outside the distraction plate were projected parallel to the central sections, and the ROIs outlined using the same reference lines.

Table 1 .
Periosteal and bone gene expression during latency period by device placement and healing period.Significant values are in bold.

Table 2 .
Morphometric parameters during latency period by device placement and healing period.