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The effect of mechanical stability on local vascularization and tissue differentiation in callus healing
ELSEVIER
Journal of Orthopaedic Research Journal of Orthopaedic Research 20 (2002) 1099-1 105 www.elsevier.coin/locaie/orthres
The effect of mechanical stability on local vascularization and tissue differentiation in callus healing Lutz Claes *, Kerstin Eckert-Hubner, Peter Augat Insliiute f o r Ortliopaedic Research und Biomechanics, Uniiwsity of lllm. Helmkoltxrrujk 14, 89081 Ulin, Gcrtiitln!,
Accepted 6 March 2002
Abstract
To investigate the influence of the stability of an osteotomy fixation on the local vascularization and tissue differentiation in callus healing, a transverse osteotomy of the right metatarsal with a gap size of 2 mm was performed in 10 sheep and stabilized with an external fixator. This fixator permitted a defined axial movement. Two groups of 5 sheep were each operated upon to allow 0.2 mm (group A) or 1 mm (group B) of axial movement. Nine weeks after surgery, the callus was dissected and histological sections prepared. The type of tissue and the vessel distribution were determined. Larger interfragmentary movements led to significantly more fibrocartilage (small axial movement A: 6.2%, large axial movement B: 21.6%) and significantly less bone formation (A: 38.2%, B: 26.3%). On average, and particularly close to the periosteum the number of vessels in the callus healing area was greater in the group with smaller movements than in the group with larger movements. There was a significant difference between the distribution of small (<20 Fin) and large (>40 pm) vessels across the whole healing area for both groups. Whereas the large vessels showed maximum density in the medullary cavity, the small vessels showed the highest frequency in the peripheral part of the periosteal callus. 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Introduction Fracture healing requires two major prerequisites, sufficient blood supply and mechanical stability. Insufficient blood supply is likely to result in a delayed union or even an atrophic non-union [14]. However, even a well-vascularized fracture healing zone will lead to a hypertrophic non-union if the mechanical stability is insufficient [13,14]. There are no studies known to us which quantitatively correlate the tissue transformation and revascularization under well-defined biomechanical conditions. However, there are some studies which show the general effect of larger interfragmentary motions on the revascularization. In experimental studies on fracture healing in canine tibiae Rhinelander demonstrated different patterns of vascularization under stable and unstable fixation conditions [ 121. He speculated, that under unstable fixation, capillaries required for osseous repair were constantly ruptured and delayed the fracture healing process resulting in the development of fibro*Corresponding author. Tel.: +49-731-500-23481; fax: +49-731-50023498. E-mail uddress: [email protected] (L. Claes).
cartilaginous tissue [12]. Since these early important investigations it is well-accepted that instability in the fracture healing zone prevents vascularization in the area of callus mineralization and leads to fibrocartilaginous tissue in the fracture gap [13,14]. While we know that small interfragmentary movements (IFMs) are benifical to fracture healing [4,5,9] and large IFMs [5,9] are critical, we know only little about the amounts of vascularization in various mechanical situations. The deposition of fibrocartilage may partially result from insufficient blood supply whereas bone formation indicates adequate vascularization under stable conditions [3,6]. Apart from fibrocartilage and bone, connective tissue is found in the callus area. Connective tissue typically occurs under adequate vascularization but serious tissue strain [3,6]. So far, little is known about the influence of the mechanical stability on the vascularization of this tissue. Few data quantitatively describe the relationship between the degree of instability (expressed as IFM or interfragmentary strain (IFS)) and the amount of blood supply found in the various tissues. Wallace et al. [I51 investigated blood supply in an osteotomy model of the
0736-0266/02/$ - see front matter 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd All rights reserved PII: SO7 3 6- 0 2 66 ( 0 2 ) 0 0 0 4 4 - X
ovine tibia using radioactive microspheres. They showed that, in the early healing phase, larger IFMs led to more corticomedullary blood supply, than smaller IFMs. After six weeks, however an approximately 50‘% higher blood supply was found in the periosteal callus for the more stable group [15]. Based on a fracture healing model with defined IFMs [4] we studied the vascularization and tissue differentiation with respect to the mechanical stability of the fracture. Our aim was to address the following questions: is there an effect of IFM on tissue differentiation; the distribution of blood vessels within the callus area; and the size of the vessels? Accordingly, to answer these questions, we performed experiments specifically to determine the sizes and distribution of blood vessels in histological sections of the healing sheep metatarsal stabilized under controlled mechanical conditions.
Materials and methods A transverse osteotomy of the right metatarsus in sheep was chosen as a standardized fracture model [1,5]. The osteotomy was stabilized with an external ring fixator that was adjustable for gap size and axial IFM (Fig. I ) . For this experiment, the external fixator was designed to provide extremely high bending and torsional stiffness while allowing axial movement through a telescoping system [4] even under low loads. Weight bearing in the operated limb produced an axial telescoping corresponding to a controlled IFM. In the unloaded swing phase of the limb, the original gap size was recovered by the recoil of the springs included in the telescoping system. A detailed description of the fixator appeared elsewhere [4]. Ten sheep that had reached full skeletal maturity (average age: 2 years; average weight: 66 kg) were investigated. This study was licensed by the government review board (Regierungsprasidium Tubingen, no. 407). The sheep were divided into two groups with osteotomy gaps or 2 mm but different I F M (group A : n = 5 , IFM = 0.2 mni; group B: n 5 , IFM = 1 mm). Nine weeks postoperatively, the sheep were sacrificed. The right metatarsals were explanted and the ring-fixators removed. The central longitudinal section of the osteotomized area was prepared for undecalcified bone histology. A central dorsoplantar longitudinal section through the callus was made with a diamond coated saw (Exakt, Norderstedt, Germany). The sections were fixed in 4% formalin solu7:
tion (pH 7.4) for a t least 48 h. They were then dehydrated in an ascending ethanol series. The dehydrated sections were embedded in methylmethacrylat (Technovit VL 7200@. Kulzer, Wertheim, Germany). After polymerisation a 70 pm slice was polished (ExaktMikroschleifsystem; Exakt, Norderstedt, Germany) and the surface stained (Fig. 2a) with Paragon (Toluidin blue and Fuchsin). The region of interest (ROI) was the callus healing area (Fig. 2b) defined by the outer diameter of the callus in radial direction and extended 2 mm proximally and distally to the centre of the osteotomy gap (Fig. 2b). The whole R 0 1 was measured using a digital caliper (Digit-Cal 5P 118704; Tesa, Switzerland) and was subdivided into nine zones (Fig. 2b). The periosteal callus (plantar and dorsal) was divided into three zones, periosteal callus peripheral (P,), periosteal callus central (P,) and periosteal callus of the middle zone (P,”). T o standardize these zones for callus formations of different sizes, the radial diameters of the zones were defined as ratio of the dilrerence between outer callus diameter and cortical (periosteal) diameter. f,,and P, were defined as 1/4 and P,, 1/2 of the outer callus (Fig. 2b). The remaining zones were the cortical gap zone (C, plantar and dorsal) and the medullary callus zone ( M ) . For the cortical gap zone ( C )the ROI extended only 1 mm distally and proximally, representing the gap area. The area covered by fibrocartilage or bone was determined using light microscopy (Axiophot; Zeiss, Oberkochen, Germany) with a net ocular (100-fold magnification) and a digital caliper (Digit-Cal 5P 1 18704; Tesa, Switzerland). The amount of new bone formation and fibrocartilage was expressed in percent with reference to the size of each individual zone. The percentage of connective tissue yielded: connective tissue (‘X)) = I OO‘%+new bone formation (‘X,)-fibrocartilage (‘%I). For the determination of blood vessel distribution all sections were evaluated under light microscopy (Olympus BX60; Olympus Optical Co. GmbH, Hamburg, Germany) with 200-fold magnification. The number and sizes (small: <20 pm; medium: 20-40 pm; large >40 pm) of the blood vessels in each type of tissue (bone, fibrocartilage or connective tissue) were determined in the different zones (Fig. 3). The largest diameter perpendicular to the longitudinal axis of the vessel visible in histological section was measured (Fig. 3). The measurement of the diameter and not the determination of the area of the vessel was chosen because the vessels were not all oriented in the same direction. For the same vessel diameter various areas would be measured depending on the angle under which the vessel was cut in the histological section. All data are repoi-ted as m e a n 5 S D . An analysis of variance (ANOVA) was performed to compare the groups with regard to statistical significant differences between groups and the location of the ROI. For the test between two groups, the Student’s I-test was used.
Results Daily observation did not indicate any difference between the two groups with regard to weight bearing of the operated limb, which returned to normal after two weeks. None of the sheep showed signs of complications and all sheep showed callus formation with peripheral bridging of the osteotomy gap. The postoperative control of the gap size and IFM produced the following results: group A: gap size 1.9 mm, IFM 0.2 mm; group B: gap size 2.1 mm, IFM 0.98 mm. Tissue diflkren tiut ion
Fig. I . Sheep metatarsal with a custom-made ring fixator. The telescoping rods connecting the proximal and distal ring allow the adjustment of initial axial movements under leg loading. The electronic measuring device (LVDT) monitors the IFM.
The amount of bone in the whole ROI was significantly larger for group A with smaller IFM than for group B ( p = 0.002, Table 1). Group A showed significantly smaller areas of fibrocartilage (p = 0.0001,
Fig. 2. (a) Histological longitudinal section through a metatarsal of group A with a callus healing (magnification five times, Paragon staining). (b) Schematic representation of the callus healing zones, M: medullary cavity, C: cortical osteotomy gap, P,: periosteal callus+entral. P,,,: periosteal callus-middle, P,,: periosteal callus-peripheral.
Table 1) than group B (Table 1). The percentage of connective tissue was almost identical for both groups (Table 1). There was a significant (p = 0.0001) influence of the localization on the new bone formation. Across the healing area, the largest percentage of new bone formation was found adjacent to the periosteum (Pc),in the middle of the periosteal callus ( P , ) and in the medullary cavity (M, Fig. 4a, Table 1). The percentage of new bone formation was smallest in the cortical gap zone (C) and in the most peripheral zone of the callus (Pp, Fig. 4a, Table 1). In all zones except the medullary cavity, bone formation was significantly (p = 0.002) smaller in group B than in group A (Fig. 4a, Table I). The relative amount of fibrocartilage was smallest in the medullary cavity (M) with relatively large values in the cortical gap (C) (Fig. 4b). For all zones, more fibrocartilage was found for group B than for group A (p = 0.0001, Fig. 4b, Table I). Irrespective of the mechanical conditions in the fracture gap there was a high percentage of connective tissue in all zones (42-72%). The highest percentage of connective tissue was found in the cortical gap (C, Table I).
formed bone the number of vessels varied from 1.3 to 8.7 per nim2 depending on the location (Table 2). The number of vessels in connective tissue varied from 0.3 to 2.7 vessels/mm2 (Table 2). Averaged over all types of tissue most of the vessels were found in the periosteal callus area (Fig. 5, Table 2) and particularly in the zones close to the periosteum (Pc).For this area significantly (p = 0.036) more vessels were seen in group A (Fig. 5). Size (fhloou' vessels
The distribution of small and large vessels showed a characteristic pattern (p = 0.0001): whereas the large vessels (>40 pm) showed their maximum occurrence in the medullary cavity (Fig. 6, Table 3); the small vessels (<20 pm) showed the highest frequency in the periosteal callus, particularly in the most peripheral part (P, and P,,, Fig. 6, Table 3). The vessels of intermediate diameter (20-40 pm) were evenly distributed across the healing area (Table 3).
Distribution of' blood vessels
Discussion
The average number of blood vessels was different for the various tissues found in the healing area. No vessels at all could be found in fibrocartilage tissue. In newly
This study quantitatively described the interaction between movement in the osteotomy gap and the distribution of newly formed blood vessels and the specific
L Cltzes r t 01. I Jourrztrl of Orthopurdic Research 20 i2002) IO99-11O5
1102
IFS = IFM divided by the fracture gap size in unloaded condition [I 11 does not exactly describe the tissue strain at various locations of the fracture gap [7,8] it allows the approximate comparison of both studies. In our experiment the initial IFS was 9% for group A (0.2 mm: (1.9 0.2) mm) and 32% for group B (0.98 mm: (2.1 0.98) mm). We found a significantly higher percentage of new bone in the healing area for group A with 9% IFS than for group B with 32% IFS and significantly less fibrocartilage in group A than in group B. The reason for finding both types of tissue for both levels of overall IFS (9% and 32'Yn) is that a certain amount of overall IFS may lead to smaller or greater tissue strains locally, depending on the specific location in the fracture gap [3,6-81. In addition, this definition of IFS is only valid at the beginning of the experiment. During the healing process the initially allowed IFM is reduced by callus formation [5]. In the periosteal ROI close to the cortex (P,) the vessel density was more than twice as large as in the medullary ROI or in the peripheral regions of the callus (Fig. 5). Moreover, in this region (Pc)the vessel density appeared to be influenced by IFM with a larger IFM resulting in a reduced number of vessels (Fig. 5). This effect is mainly caused by the larger amount of nonvascularized fibrocartilage seen in this area when a larger IFM was allowed (Table 1). The areal density of blood vessels in the mineralized callus seems to be independent of the IFM. This can be explained by the local mechanical conditions of these vessels. They are mechanically protected by the surrounding mineralized trabecular structure of the bony callus and are not subjected to the IFS which mainly takes place in the non-mineralized part of the callus [6,7]. The influence of the IFM on the vascularization of the callus area seems to be different in the early and late healing phase, as shown by Wallace et al. [15]. Whereas in the early healing phase, more IFM seems to promote vascularization in the later phase the more stable fixation seems to be better. However the comparison of these two studies is limited because Wallace et al. mea-
+ +
Fig. 3. Characteristical histological section of callus tissue (magnification 200x). Calcified callus with active osteoblasts and vessels of large (L), medium ( M ) and small (s) diameter in between the bony trabeculae (Paragon staining).
tissue formation. More axial IFM led to less bone formation in the healing zone and more fibrocartilage. The decreasing amount of bone and increasing amount of fibrocartilage in the osteotomy gap with higher IFS is consistent with former studies conducted by our group [l], and with the experimental results of Hente et al. [lo]. Hente et al. created a sheep model with a 2 mm gap and a gradient of IFS ranging from 0% to 50%. They found bony bridging for strains < lo%, no new bone formation for strains > 30% and no vascularity for strains > 40%. Even though the IFS definition
Table 1 Tissue distribution ('%I)
Bone
A B
3 1 . 4 f 19.6 48.4*49.3 10.8i7.0 31.2i13.6
44.6i20.6 32.0315.2
24.0* 13.7 4 8 . 2 i 2 0 . 9 1.6*18.2 50.4f13.5
19.6f30.9 43.0f7.1 0.8f21.3 37.0f10.2
51.2f6.6 41.4f10.4
34.056.2 31.2f9.9
Fibrocartilage
A
7.4i3.8
9.256.5
6.8f8.7
5.6f5.0
5.0i5.8
8.0i8.9
3.8f3.4
6.8f6.6
2.8f3.9
B
38.0526.9
22.0f17.7
22.6f12.9
31.6f26.4
11.4i6.9
30.4Xt22.6
12.6f10.4
16.2i14.1
9.4h6.7
A B
61.2f19.7 51.2i23.3
4 2 . 4 h 10.4 47.0f21.6 4 6 . 8 f l l . l 43.4517.0
72.4f31.6 68.8f22.9
53.2f6.2 51.2111.2
4 2 . 0 4 ~10.5 6 1 . 2 i 17.0 41.6~k15.4 59.4f13.5
Connective tissue
70.4zk 16.5 4 6 . 8 f 2 3 . 7 66.8i20.9 32.4i12.1
~~~
Mean values f SD. Left to right columns represent locations from plantar to dorsal.
I I03
60
40 -Group
A 141~1
30
-
t
--t-G~roup 4-Group
i
A
\
\ 20
L-4
/ ‘
A
B
A
‘
/ \ I
\
/
\ \
k4,
\ ‘I/ 10
O
b)
a)
Fig. 4. Frequency distribution of bone (a) and fibrocartilage (b) found in the various healing zones
Table 2 Number of blood vesselslmm’
Bone
Group
Pp
pln
PC
C
M
C
PC
p r n
PP
A
4.0f1.6 8.7fS.4
4.lf1.9 8.4f7.0
7.Sf4.6 4.8f2.3
1.8k0.7 1.4i0.9
2.0f1.8 1.5+1.1
1.3f0.8 1.6f1.4
2.3f2.S 4.0f3.1
2.111.9 3.052.0
1.S3Z2.4 2.0311.2
1.7f1.8 2.3f2.3
1.8k1.7 1.8hl.S
1.2f1.2 l.OiO.8
2.7f2.3 1.8f1.3
2.4t1.8 1.9hI.5
1.3f0.7 1.451.2
1.9h2.1 l.OkO.7
1.0310.6 0.8+0.8
0.Sf0.3 0.350.3
B Connective tissue
A
B
Mean values iSD. Left to right columns represent locations from plantar to dorsal.
4 -+--Group
A
[1/rnm2 3
2
1
0
Fig. 5 . Distribution of the vesselslmm’ for the various healing zones for all types of tissue together.
sured blood flow in a bone volume of 10 mm proximally and distally to the osteotomy gap whereas we determined the number of vessels in one longitudinal section only in the zone of 2 mm proximally and distally to the osteotomy line. The progression of callus formation by endochondral ossification occurs at the borderline between hard callus and soft callus. Whether the fracture callus will achieve bony bridging or a layer of fibrocartilage remains depends on the vascularization and the mechanical situation at this front of callus mineralization. Despite sufficient blood supply in the mineralized callus hypertrophic non-unions fail to heal because a non-vascularized layer of fibrocartilage remains [14]. Therefore, our measurement of vascularization was not based on the overall callus volume but was focused on a ROI at the mineralization front. There was a characteristic vessel distribution across the fracture callus which was similar for group A and group B. The larger vessels were found in the centre and the smaller ones more in the periphery of the periosteal callus (Fig. 6). By the time of our investigation (at 9
I I04
L. Clues
1'1
ul. 1 Journal oJ' Orthipuixliir Re.~c~urc/z 20 (2002J 1099-//05
["/.I
40
20
i
< 20 pm
0
Fig. 6. Frequency distribution of large (>40 pin) and small (<20 pm) vessels in new bone formation and connective tissue for the various healing mnes and group A and group B.
weeks) the medullary cavity had already been revascularized [12,13], with vessels of large diameter. This is the typical image of a revascularization of an undisplaced fracture [ 121. At first sight it might be surprising that the vessel distribution across the callus was not symmetrical (Fig. 5 ) even though a symmetrical external fixator was chosen. The reason might be the asymmetrical arrangement of blood vessels and tendons on the dorsal and plantar aspect of the metatarsal. That is, the large extensor tendon in close contact to the plantar surface of the metatarsal limits the extension of the callus and causes an asymmetrical callus formation. Our statements regarding the correlations between the stability (IFM or IFS) and the tissue differentiation and vascularization are limited because only two bio-
mechanical conditions were investigated. Both groups developed significantly different amounts of well-vascularized bone and non-vascularized fibrocartilage but nevertheless showed signs of bony bridging by the termination of the experiment. One of the reasons for this finding could be that the critical overall IFS for the fracture healing process might be above 40% as mentioned by Hente et al. [lo] and the IFS in both groups were below this level. It is hardly possible to define a numerical borderline which decides whether vascularized connective tissue or non-vascularized fibrocartilage develops. Besides the amount of tissue strain, the level of hydrostatic pressure in the tissue may play a role [2,6,7]. Neither the local tissue strain nor the hydrostatic pressure are known for our experimental situation. One may speculate that large tissue strains can cause a rupture of the vascularizing vessels [I31 but also that a hydrostatic pressure which is too high results in the collapse of vessels and prevents their propagation [3]. However, as long as the IFS strain is not too high the fibrocartilage can lead to callus healing by endochondral ossification as demonstrated for the metatarsals of group B. The data were based on measurements of one histological longitudinal section, and it was assumed that this represents the tissue and vascular distribution at the fracture site. At the moment there is no method available to us which would allow a fully 3D-reconstruction of tissue and vessel distribution. The data presented in this study concerning the vascularizatioii of the various callus tissues permits an estimation of the vascularization of the fracture callus. Blood supply correlates positively with the amount of bone formation and negatively with the amount of fibrocartilage. We found a similar correlation for the biomechanical strength of the callus. Our group [ I ] has already demonstrated that the percentage of bone in the fracture gap correlated significantly with the bending stiffness (R' = 0.8). In the current study group A with more bone and less fibrocartilage in the fracture gap showed a significantly higher bending stiffness (group A: 28 Nm/mm, group B: 20 Nm/mm) as reported previously [5].
Table 3 Vessel distribution ('XI) ~
~~
~~
~~
~~~
48.2i23.5 5 0 . 6 3 ~14.7
3 1 . 2 f 19.2 3 2 . 8 3 ~13.1
26.4i8.7 34.2f20.2
25.2i11.0 19.2f8.2
20.455.7 13.6i40.3
26.6i9.8 1 7 . 2 i 13.8
13.8f16.8 20.03~15.4
21.0f8.3 31.4f9.0
25.8i31.6 5 4 . 4 3 ~1.5.4
B
39.6i15.4 44.0515.6
38.4i6.0 49.8+5.0
35.6i6.3 42.4f13.0
38.8f5.0 46.8+11.8
34.0i1.1 38.4+9.3
44.2f6.5 39.8f10.5
55.4+16.7 46.6f10.1
44.6312.1 41.25k4.3
47.2+27.8 36.0515.1
A B
12.2i12.1 5.4f7.0
30.4f14.7 17.4i9.3
37.8f11.8 2 3 . 4 f 16.0
36.0f0.8 34.0i- 12.9
45.6i6.2 48.0% 11.9
29.2i11.3 43.0% 16.7
30.8*7.6 3 3 . 4 4 ~19.7
34.4i13.5 2 7 . 4 i 10.6
9.6k9.6
Small vessels
A
Middle vessels
A
Large vessels
B
Mean values f SD. Left to right columns represent locations from plantar to dorsal.
27.0i30.3
L. Clues et 01. I Journul of Orthopnctlic Rcwcirch 20 (2002) 1099-1 105
To our knowledge, this is the first study which presents quantitative data about the vascularization in the healing callus. Even though the data are limited and the number of animals was small, we found some statistically significant differences which indicate a strong association between fracture stability and the spatial distribution of newly formed blood vessels and specific tissue formation. From a clinical point of view these results indicate the important role of stability for the tissue differentiation and revascularization of fractures in the late phase of fracture healing.
Acknowledgement
This study was kindly supported by the German Research Council (DFG-Cl 77/2).
References [ I ] Augat P, Margevicius K, Simon J, Wolf S, Suger G, Claes L. Influence of size and stability of the osteotomy gap. J Orthop Res 1998;16:475-81, [2] Carter DR, Blenman PR, Beaupre GS. Correlations between mechanical stress history and tissue differentiation in initial fracture healing. J Orthop Res I988;6:73648. [3] Carter DR, Beauprt GS, Giori NJ, Helms DDS. Mechanobiology of skeletal regeneration. Clin Orthop Re1 Res 1998;355S:41-55.
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[4] Claes L, Wilke HJ, Augat P, Rubenacker S, Margevicius KJ. Effect of dynamisation on gap healing of diaphyseal fractures under external fixation. Clin Biomech 1995;10(5):227-34. [5] Claes L, Augat P, Suger G, Wilke HJ. Influence of size and stability of the osteotomy gap on the success of fracture healing. J Orthop Res 1997;15:577-84. [6] Claes LE, Heigele CA, Neidlinger-Wilke C, Kaspar D. Seidl W, Margevicius KJ, Augat P. Effects of mechanical factors 011 the fracture healing process. Clin Orthop Re1 Res 1998;355S:132- 47. [7] Claes LE, Heigele CA. Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing. J Biomech 1999;32:255-66. [8] Di Gioia AMI, Chcal EJ, Hayes WC. Three-dimensional strain fields in an uniform osteotomy gap. J Biomech Eng 1986:108:27380. [9] Goodship AE, Kenwright J. The influence of induced micromovement upon the healing of experimental fractures. J Bone Joint Surg 1993;67B:650-5. [lo] Hente R, Cheal EJ, Perren SM. Die dehnungstheorie als erklarungsgrundlage des erfolges der biologischen osteosynthese. Hefte zu Der Unfallchirurg 1992;232:445-7. [l I ] Perren SM, Cordey J. The concept of interfragmentary strain. In: Uthoff H K , editor. Current Concepts of Internal Fixation of Fractures. Berlin: Springer; 1980. p. 63-77. [12] Rhinelander FW. Tibia1 blood supply in relation to fracture healing. Clin Orthop 1974;105:34 81. [I31 Schenk RK, Willenegger H. Zur Histologie der primiren Knochenheilung. Unfallheilkunde 1977;80: 155-60. [I41 Schweiberer L, Schenk R . Histomorphologie und Vaskularisation der sekundaren Knochenbruchheilung unter besonderer Beriicksichtigung der Tibiaschaftfraktur. Unfallheilkunde 1977: 80:275-86. [15] Wallace AL, Draper ER. Stracham RK, McCarthy ID, Hughes SPF. The vascular response to fracture micromovement. Clin Orthop Re1 Res 1994;301:281-90.
Journal of Orthopaedic Research Journal of Orthopaedic Research 20 (2002) 1099-1 105 www.elsevier.coin/locaie/orthres
The effect of mechanical stability on local vascularization and tissue differentiation in callus healing Lutz Claes *, Kerstin Eckert-Hubner, Peter Augat Insliiute f o r Ortliopaedic Research und Biomechanics, Uniiwsity of lllm. Helmkoltxrrujk 14, 89081 Ulin, Gcrtiitln!,
Accepted 6 March 2002
Abstract
To investigate the influence of the stability of an osteotomy fixation on the local vascularization and tissue differentiation in callus healing, a transverse osteotomy of the right metatarsal with a gap size of 2 mm was performed in 10 sheep and stabilized with an external fixator. This fixator permitted a defined axial movement. Two groups of 5 sheep were each operated upon to allow 0.2 mm (group A) or 1 mm (group B) of axial movement. Nine weeks after surgery, the callus was dissected and histological sections prepared. The type of tissue and the vessel distribution were determined. Larger interfragmentary movements led to significantly more fibrocartilage (small axial movement A: 6.2%, large axial movement B: 21.6%) and significantly less bone formation (A: 38.2%, B: 26.3%). On average, and particularly close to the periosteum the number of vessels in the callus healing area was greater in the group with smaller movements than in the group with larger movements. There was a significant difference between the distribution of small (<20 Fin) and large (>40 pm) vessels across the whole healing area for both groups. Whereas the large vessels showed maximum density in the medullary cavity, the small vessels showed the highest frequency in the peripheral part of the periosteal callus. 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Introduction Fracture healing requires two major prerequisites, sufficient blood supply and mechanical stability. Insufficient blood supply is likely to result in a delayed union or even an atrophic non-union [14]. However, even a well-vascularized fracture healing zone will lead to a hypertrophic non-union if the mechanical stability is insufficient [13,14]. There are no studies known to us which quantitatively correlate the tissue transformation and revascularization under well-defined biomechanical conditions. However, there are some studies which show the general effect of larger interfragmentary motions on the revascularization. In experimental studies on fracture healing in canine tibiae Rhinelander demonstrated different patterns of vascularization under stable and unstable fixation conditions [ 121. He speculated, that under unstable fixation, capillaries required for osseous repair were constantly ruptured and delayed the fracture healing process resulting in the development of fibro*Corresponding author. Tel.: +49-731-500-23481; fax: +49-731-50023498. E-mail uddress: [email protected] (L. Claes).
cartilaginous tissue [12]. Since these early important investigations it is well-accepted that instability in the fracture healing zone prevents vascularization in the area of callus mineralization and leads to fibrocartilaginous tissue in the fracture gap [13,14]. While we know that small interfragmentary movements (IFMs) are benifical to fracture healing [4,5,9] and large IFMs [5,9] are critical, we know only little about the amounts of vascularization in various mechanical situations. The deposition of fibrocartilage may partially result from insufficient blood supply whereas bone formation indicates adequate vascularization under stable conditions [3,6]. Apart from fibrocartilage and bone, connective tissue is found in the callus area. Connective tissue typically occurs under adequate vascularization but serious tissue strain [3,6]. So far, little is known about the influence of the mechanical stability on the vascularization of this tissue. Few data quantitatively describe the relationship between the degree of instability (expressed as IFM or interfragmentary strain (IFS)) and the amount of blood supply found in the various tissues. Wallace et al. [I51 investigated blood supply in an osteotomy model of the
0736-0266/02/$ - see front matter 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd All rights reserved PII: SO7 3 6- 0 2 66 ( 0 2 ) 0 0 0 4 4 - X
ovine tibia using radioactive microspheres. They showed that, in the early healing phase, larger IFMs led to more corticomedullary blood supply, than smaller IFMs. After six weeks, however an approximately 50‘% higher blood supply was found in the periosteal callus for the more stable group [15]. Based on a fracture healing model with defined IFMs [4] we studied the vascularization and tissue differentiation with respect to the mechanical stability of the fracture. Our aim was to address the following questions: is there an effect of IFM on tissue differentiation; the distribution of blood vessels within the callus area; and the size of the vessels? Accordingly, to answer these questions, we performed experiments specifically to determine the sizes and distribution of blood vessels in histological sections of the healing sheep metatarsal stabilized under controlled mechanical conditions.
Materials and methods A transverse osteotomy of the right metatarsus in sheep was chosen as a standardized fracture model [1,5]. The osteotomy was stabilized with an external ring fixator that was adjustable for gap size and axial IFM (Fig. I ) . For this experiment, the external fixator was designed to provide extremely high bending and torsional stiffness while allowing axial movement through a telescoping system [4] even under low loads. Weight bearing in the operated limb produced an axial telescoping corresponding to a controlled IFM. In the unloaded swing phase of the limb, the original gap size was recovered by the recoil of the springs included in the telescoping system. A detailed description of the fixator appeared elsewhere [4]. Ten sheep that had reached full skeletal maturity (average age: 2 years; average weight: 66 kg) were investigated. This study was licensed by the government review board (Regierungsprasidium Tubingen, no. 407). The sheep were divided into two groups with osteotomy gaps or 2 mm but different I F M (group A : n = 5 , IFM = 0.2 mni; group B: n 5 , IFM = 1 mm). Nine weeks postoperatively, the sheep were sacrificed. The right metatarsals were explanted and the ring-fixators removed. The central longitudinal section of the osteotomized area was prepared for undecalcified bone histology. A central dorsoplantar longitudinal section through the callus was made with a diamond coated saw (Exakt, Norderstedt, Germany). The sections were fixed in 4% formalin solu7:
tion (pH 7.4) for a t least 48 h. They were then dehydrated in an ascending ethanol series. The dehydrated sections were embedded in methylmethacrylat (Technovit VL 7200@. Kulzer, Wertheim, Germany). After polymerisation a 70 pm slice was polished (ExaktMikroschleifsystem; Exakt, Norderstedt, Germany) and the surface stained (Fig. 2a) with Paragon (Toluidin blue and Fuchsin). The region of interest (ROI) was the callus healing area (Fig. 2b) defined by the outer diameter of the callus in radial direction and extended 2 mm proximally and distally to the centre of the osteotomy gap (Fig. 2b). The whole R 0 1 was measured using a digital caliper (Digit-Cal 5P 118704; Tesa, Switzerland) and was subdivided into nine zones (Fig. 2b). The periosteal callus (plantar and dorsal) was divided into three zones, periosteal callus peripheral (P,), periosteal callus central (P,) and periosteal callus of the middle zone (P,”). T o standardize these zones for callus formations of different sizes, the radial diameters of the zones were defined as ratio of the dilrerence between outer callus diameter and cortical (periosteal) diameter. f,,and P, were defined as 1/4 and P,, 1/2 of the outer callus (Fig. 2b). The remaining zones were the cortical gap zone (C, plantar and dorsal) and the medullary callus zone ( M ) . For the cortical gap zone ( C )the ROI extended only 1 mm distally and proximally, representing the gap area. The area covered by fibrocartilage or bone was determined using light microscopy (Axiophot; Zeiss, Oberkochen, Germany) with a net ocular (100-fold magnification) and a digital caliper (Digit-Cal 5P 1 18704; Tesa, Switzerland). The amount of new bone formation and fibrocartilage was expressed in percent with reference to the size of each individual zone. The percentage of connective tissue yielded: connective tissue (‘X)) = I OO‘%+new bone formation (‘X,)-fibrocartilage (‘%I). For the determination of blood vessel distribution all sections were evaluated under light microscopy (Olympus BX60; Olympus Optical Co. GmbH, Hamburg, Germany) with 200-fold magnification. The number and sizes (small: <20 pm; medium: 20-40 pm; large >40 pm) of the blood vessels in each type of tissue (bone, fibrocartilage or connective tissue) were determined in the different zones (Fig. 3). The largest diameter perpendicular to the longitudinal axis of the vessel visible in histological section was measured (Fig. 3). The measurement of the diameter and not the determination of the area of the vessel was chosen because the vessels were not all oriented in the same direction. For the same vessel diameter various areas would be measured depending on the angle under which the vessel was cut in the histological section. All data are repoi-ted as m e a n 5 S D . An analysis of variance (ANOVA) was performed to compare the groups with regard to statistical significant differences between groups and the location of the ROI. For the test between two groups, the Student’s I-test was used.
Results Daily observation did not indicate any difference between the two groups with regard to weight bearing of the operated limb, which returned to normal after two weeks. None of the sheep showed signs of complications and all sheep showed callus formation with peripheral bridging of the osteotomy gap. The postoperative control of the gap size and IFM produced the following results: group A: gap size 1.9 mm, IFM 0.2 mm; group B: gap size 2.1 mm, IFM 0.98 mm. Tissue diflkren tiut ion
Fig. I . Sheep metatarsal with a custom-made ring fixator. The telescoping rods connecting the proximal and distal ring allow the adjustment of initial axial movements under leg loading. The electronic measuring device (LVDT) monitors the IFM.
The amount of bone in the whole ROI was significantly larger for group A with smaller IFM than for group B ( p = 0.002, Table 1). Group A showed significantly smaller areas of fibrocartilage (p = 0.0001,
Fig. 2. (a) Histological longitudinal section through a metatarsal of group A with a callus healing (magnification five times, Paragon staining). (b) Schematic representation of the callus healing zones, M: medullary cavity, C: cortical osteotomy gap, P,: periosteal callus+entral. P,,,: periosteal callus-middle, P,,: periosteal callus-peripheral.
Table 1) than group B (Table 1). The percentage of connective tissue was almost identical for both groups (Table 1). There was a significant (p = 0.0001) influence of the localization on the new bone formation. Across the healing area, the largest percentage of new bone formation was found adjacent to the periosteum (Pc),in the middle of the periosteal callus ( P , ) and in the medullary cavity (M, Fig. 4a, Table 1). The percentage of new bone formation was smallest in the cortical gap zone (C) and in the most peripheral zone of the callus (Pp, Fig. 4a, Table 1). In all zones except the medullary cavity, bone formation was significantly (p = 0.002) smaller in group B than in group A (Fig. 4a, Table I). The relative amount of fibrocartilage was smallest in the medullary cavity (M) with relatively large values in the cortical gap (C) (Fig. 4b). For all zones, more fibrocartilage was found for group B than for group A (p = 0.0001, Fig. 4b, Table I). Irrespective of the mechanical conditions in the fracture gap there was a high percentage of connective tissue in all zones (42-72%). The highest percentage of connective tissue was found in the cortical gap (C, Table I).
formed bone the number of vessels varied from 1.3 to 8.7 per nim2 depending on the location (Table 2). The number of vessels in connective tissue varied from 0.3 to 2.7 vessels/mm2 (Table 2). Averaged over all types of tissue most of the vessels were found in the periosteal callus area (Fig. 5, Table 2) and particularly in the zones close to the periosteum (Pc).For this area significantly (p = 0.036) more vessels were seen in group A (Fig. 5). Size (fhloou' vessels
The distribution of small and large vessels showed a characteristic pattern (p = 0.0001): whereas the large vessels (>40 pm) showed their maximum occurrence in the medullary cavity (Fig. 6, Table 3); the small vessels (<20 pm) showed the highest frequency in the periosteal callus, particularly in the most peripheral part (P, and P,,, Fig. 6, Table 3). The vessels of intermediate diameter (20-40 pm) were evenly distributed across the healing area (Table 3).
Distribution of' blood vessels
Discussion
The average number of blood vessels was different for the various tissues found in the healing area. No vessels at all could be found in fibrocartilage tissue. In newly
This study quantitatively described the interaction between movement in the osteotomy gap and the distribution of newly formed blood vessels and the specific
L Cltzes r t 01. I Jourrztrl of Orthopurdic Research 20 i2002) IO99-11O5
1102
IFS = IFM divided by the fracture gap size in unloaded condition [I 11 does not exactly describe the tissue strain at various locations of the fracture gap [7,8] it allows the approximate comparison of both studies. In our experiment the initial IFS was 9% for group A (0.2 mm: (1.9 0.2) mm) and 32% for group B (0.98 mm: (2.1 0.98) mm). We found a significantly higher percentage of new bone in the healing area for group A with 9% IFS than for group B with 32% IFS and significantly less fibrocartilage in group A than in group B. The reason for finding both types of tissue for both levels of overall IFS (9% and 32'Yn) is that a certain amount of overall IFS may lead to smaller or greater tissue strains locally, depending on the specific location in the fracture gap [3,6-81. In addition, this definition of IFS is only valid at the beginning of the experiment. During the healing process the initially allowed IFM is reduced by callus formation [5]. In the periosteal ROI close to the cortex (P,) the vessel density was more than twice as large as in the medullary ROI or in the peripheral regions of the callus (Fig. 5). Moreover, in this region (Pc)the vessel density appeared to be influenced by IFM with a larger IFM resulting in a reduced number of vessels (Fig. 5). This effect is mainly caused by the larger amount of nonvascularized fibrocartilage seen in this area when a larger IFM was allowed (Table 1). The areal density of blood vessels in the mineralized callus seems to be independent of the IFM. This can be explained by the local mechanical conditions of these vessels. They are mechanically protected by the surrounding mineralized trabecular structure of the bony callus and are not subjected to the IFS which mainly takes place in the non-mineralized part of the callus [6,7]. The influence of the IFM on the vascularization of the callus area seems to be different in the early and late healing phase, as shown by Wallace et al. [15]. Whereas in the early healing phase, more IFM seems to promote vascularization in the later phase the more stable fixation seems to be better. However the comparison of these two studies is limited because Wallace et al. mea-
+ +
Fig. 3. Characteristical histological section of callus tissue (magnification 200x). Calcified callus with active osteoblasts and vessels of large (L), medium ( M ) and small (s) diameter in between the bony trabeculae (Paragon staining).
tissue formation. More axial IFM led to less bone formation in the healing zone and more fibrocartilage. The decreasing amount of bone and increasing amount of fibrocartilage in the osteotomy gap with higher IFS is consistent with former studies conducted by our group [l], and with the experimental results of Hente et al. [lo]. Hente et al. created a sheep model with a 2 mm gap and a gradient of IFS ranging from 0% to 50%. They found bony bridging for strains < lo%, no new bone formation for strains > 30% and no vascularity for strains > 40%. Even though the IFS definition
Table 1 Tissue distribution ('%I)
Bone
A B
3 1 . 4 f 19.6 48.4*49.3 10.8i7.0 31.2i13.6
44.6i20.6 32.0315.2
24.0* 13.7 4 8 . 2 i 2 0 . 9 1.6*18.2 50.4f13.5
19.6f30.9 43.0f7.1 0.8f21.3 37.0f10.2
51.2f6.6 41.4f10.4
34.056.2 31.2f9.9
Fibrocartilage
A
7.4i3.8
9.256.5
6.8f8.7
5.6f5.0
5.0i5.8
8.0i8.9
3.8f3.4
6.8f6.6
2.8f3.9
B
38.0526.9
22.0f17.7
22.6f12.9
31.6f26.4
11.4i6.9
30.4Xt22.6
12.6f10.4
16.2i14.1
9.4h6.7
A B
61.2f19.7 51.2i23.3
4 2 . 4 h 10.4 47.0f21.6 4 6 . 8 f l l . l 43.4517.0
72.4f31.6 68.8f22.9
53.2f6.2 51.2111.2
4 2 . 0 4 ~10.5 6 1 . 2 i 17.0 41.6~k15.4 59.4f13.5
Connective tissue
70.4zk 16.5 4 6 . 8 f 2 3 . 7 66.8i20.9 32.4i12.1
~~~
Mean values f SD. Left to right columns represent locations from plantar to dorsal.
I I03
60
40 -Group
A 141~1
30
-
t
--t-G~roup 4-Group
i
A
\
\ 20
L-4
/ ‘
A
B
A
‘
/ \ I
\
/
\ \
k4,
\ ‘I/ 10
O
b)
a)
Fig. 4. Frequency distribution of bone (a) and fibrocartilage (b) found in the various healing zones
Table 2 Number of blood vesselslmm’
Bone
Group
Pp
pln
PC
C
M
C
PC
p r n
PP
A
4.0f1.6 8.7fS.4
4.lf1.9 8.4f7.0
7.Sf4.6 4.8f2.3
1.8k0.7 1.4i0.9
2.0f1.8 1.5+1.1
1.3f0.8 1.6f1.4
2.3f2.S 4.0f3.1
2.111.9 3.052.0
1.S3Z2.4 2.0311.2
1.7f1.8 2.3f2.3
1.8k1.7 1.8hl.S
1.2f1.2 l.OiO.8
2.7f2.3 1.8f1.3
2.4t1.8 1.9hI.5
1.3f0.7 1.451.2
1.9h2.1 l.OkO.7
1.0310.6 0.8+0.8
0.Sf0.3 0.350.3
B Connective tissue
A
B
Mean values iSD. Left to right columns represent locations from plantar to dorsal.
4 -+--Group
A
[1/rnm2 3
2
1
0
Fig. 5 . Distribution of the vesselslmm’ for the various healing zones for all types of tissue together.
sured blood flow in a bone volume of 10 mm proximally and distally to the osteotomy gap whereas we determined the number of vessels in one longitudinal section only in the zone of 2 mm proximally and distally to the osteotomy line. The progression of callus formation by endochondral ossification occurs at the borderline between hard callus and soft callus. Whether the fracture callus will achieve bony bridging or a layer of fibrocartilage remains depends on the vascularization and the mechanical situation at this front of callus mineralization. Despite sufficient blood supply in the mineralized callus hypertrophic non-unions fail to heal because a non-vascularized layer of fibrocartilage remains [14]. Therefore, our measurement of vascularization was not based on the overall callus volume but was focused on a ROI at the mineralization front. There was a characteristic vessel distribution across the fracture callus which was similar for group A and group B. The larger vessels were found in the centre and the smaller ones more in the periphery of the periosteal callus (Fig. 6). By the time of our investigation (at 9
I I04
L. Clues
1'1
ul. 1 Journal oJ' Orthipuixliir Re.~c~urc/z 20 (2002J 1099-//05
["/.I
40
20
i
< 20 pm
0
Fig. 6. Frequency distribution of large (>40 pin) and small (<20 pm) vessels in new bone formation and connective tissue for the various healing mnes and group A and group B.
weeks) the medullary cavity had already been revascularized [12,13], with vessels of large diameter. This is the typical image of a revascularization of an undisplaced fracture [ 121. At first sight it might be surprising that the vessel distribution across the callus was not symmetrical (Fig. 5 ) even though a symmetrical external fixator was chosen. The reason might be the asymmetrical arrangement of blood vessels and tendons on the dorsal and plantar aspect of the metatarsal. That is, the large extensor tendon in close contact to the plantar surface of the metatarsal limits the extension of the callus and causes an asymmetrical callus formation. Our statements regarding the correlations between the stability (IFM or IFS) and the tissue differentiation and vascularization are limited because only two bio-
mechanical conditions were investigated. Both groups developed significantly different amounts of well-vascularized bone and non-vascularized fibrocartilage but nevertheless showed signs of bony bridging by the termination of the experiment. One of the reasons for this finding could be that the critical overall IFS for the fracture healing process might be above 40% as mentioned by Hente et al. [lo] and the IFS in both groups were below this level. It is hardly possible to define a numerical borderline which decides whether vascularized connective tissue or non-vascularized fibrocartilage develops. Besides the amount of tissue strain, the level of hydrostatic pressure in the tissue may play a role [2,6,7]. Neither the local tissue strain nor the hydrostatic pressure are known for our experimental situation. One may speculate that large tissue strains can cause a rupture of the vascularizing vessels [I31 but also that a hydrostatic pressure which is too high results in the collapse of vessels and prevents their propagation [3]. However, as long as the IFS strain is not too high the fibrocartilage can lead to callus healing by endochondral ossification as demonstrated for the metatarsals of group B. The data were based on measurements of one histological longitudinal section, and it was assumed that this represents the tissue and vascular distribution at the fracture site. At the moment there is no method available to us which would allow a fully 3D-reconstruction of tissue and vessel distribution. The data presented in this study concerning the vascularizatioii of the various callus tissues permits an estimation of the vascularization of the fracture callus. Blood supply correlates positively with the amount of bone formation and negatively with the amount of fibrocartilage. We found a similar correlation for the biomechanical strength of the callus. Our group [ I ] has already demonstrated that the percentage of bone in the fracture gap correlated significantly with the bending stiffness (R' = 0.8). In the current study group A with more bone and less fibrocartilage in the fracture gap showed a significantly higher bending stiffness (group A: 28 Nm/mm, group B: 20 Nm/mm) as reported previously [5].
Table 3 Vessel distribution ('XI) ~
~~
~~
~~
~~~
48.2i23.5 5 0 . 6 3 ~14.7
3 1 . 2 f 19.2 3 2 . 8 3 ~13.1
26.4i8.7 34.2f20.2
25.2i11.0 19.2f8.2
20.455.7 13.6i40.3
26.6i9.8 1 7 . 2 i 13.8
13.8f16.8 20.03~15.4
21.0f8.3 31.4f9.0
25.8i31.6 5 4 . 4 3 ~1.5.4
B
39.6i15.4 44.0515.6
38.4i6.0 49.8+5.0
35.6i6.3 42.4f13.0
38.8f5.0 46.8+11.8
34.0i1.1 38.4+9.3
44.2f6.5 39.8f10.5
55.4+16.7 46.6f10.1
44.6312.1 41.25k4.3
47.2+27.8 36.0515.1
A B
12.2i12.1 5.4f7.0
30.4f14.7 17.4i9.3
37.8f11.8 2 3 . 4 f 16.0
36.0f0.8 34.0i- 12.9
45.6i6.2 48.0% 11.9
29.2i11.3 43.0% 16.7
30.8*7.6 3 3 . 4 4 ~19.7
34.4i13.5 2 7 . 4 i 10.6
9.6k9.6
Small vessels
A
Middle vessels
A
Large vessels
B
Mean values f SD. Left to right columns represent locations from plantar to dorsal.
27.0i30.3
L. Clues et 01. I Journul of Orthopnctlic Rcwcirch 20 (2002) 1099-1 105
To our knowledge, this is the first study which presents quantitative data about the vascularization in the healing callus. Even though the data are limited and the number of animals was small, we found some statistically significant differences which indicate a strong association between fracture stability and the spatial distribution of newly formed blood vessels and specific tissue formation. From a clinical point of view these results indicate the important role of stability for the tissue differentiation and revascularization of fractures in the late phase of fracture healing.
Acknowledgement
This study was kindly supported by the German Research Council (DFG-Cl 77/2).
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[4] Claes L, Wilke HJ, Augat P, Rubenacker S, Margevicius KJ. Effect of dynamisation on gap healing of diaphyseal fractures under external fixation. Clin Biomech 1995;10(5):227-34. [5] Claes L, Augat P, Suger G, Wilke HJ. Influence of size and stability of the osteotomy gap on the success of fracture healing. J Orthop Res 1997;15:577-84. [6] Claes LE, Heigele CA, Neidlinger-Wilke C, Kaspar D. Seidl W, Margevicius KJ, Augat P. Effects of mechanical factors 011 the fracture healing process. Clin Orthop Re1 Res 1998;355S:132- 47. [7] Claes LE, Heigele CA. Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing. J Biomech 1999;32:255-66. [8] Di Gioia AMI, Chcal EJ, Hayes WC. Three-dimensional strain fields in an uniform osteotomy gap. J Biomech Eng 1986:108:27380. [9] Goodship AE, Kenwright J. The influence of induced micromovement upon the healing of experimental fractures. J Bone Joint Surg 1993;67B:650-5. [lo] Hente R, Cheal EJ, Perren SM. Die dehnungstheorie als erklarungsgrundlage des erfolges der biologischen osteosynthese. Hefte zu Der Unfallchirurg 1992;232:445-7. [l I ] Perren SM, Cordey J. The concept of interfragmentary strain. In: Uthoff H K , editor. Current Concepts of Internal Fixation of Fractures. Berlin: Springer; 1980. p. 63-77. [12] Rhinelander FW. Tibia1 blood supply in relation to fracture healing. Clin Orthop 1974;105:34 81. [I31 Schenk RK, Willenegger H. Zur Histologie der primiren Knochenheilung. Unfallheilkunde 1977;80: 155-60. [I41 Schweiberer L, Schenk R . Histomorphologie und Vaskularisation der sekundaren Knochenbruchheilung unter besonderer Beriicksichtigung der Tibiaschaftfraktur. Unfallheilkunde 1977: 80:275-86. [15] Wallace AL, Draper ER. Stracham RK, McCarthy ID, Hughes SPF. The vascular response to fracture micromovement. Clin Orthop Re1 Res 1994;301:281-90.