Effect of Vitamin C on Fracture Healing in Elderly Osteogenic Disorder Shionogi Rats

By Alcantara-Martos, T; Delgado-Martinez, A D; Vega, M V; Carrascal, M T; Munuera-Martinez, L

We studied the effect of vitamin C on fracture healing in the elderly. A total of 80 elderly Osteogenic Disorder Shionogi rats were divided into four groups with different rates of vitamin C intake. A closed bilateral fracture was made in the middle third of the femur of each rat. Five weeks after fracture the femora were analysed by mechanical and histological testing. The groups with the lower vitamin C intake demonstrated a lower mechanical resistance of the healing callus and a lower histological grade. The vitamin C levels in blood during healing correlated with the torque resistance of the callus formed (r = 0.525). Therefore, the supplementary vitamin C improved the mechanical resistance of the fracture callus in elderly rats. If these results are similar in humans, vitamin C supplementation should be recommended during fracture healing in the elderly.

Vitamin C is a key element in the healing of fractures. It functions in the hydroxylan’on of two amino acids (proline and lysine) needed to form the triple helix of collagen,1 which is essential for the formation of immature callus in bone healing.2 Vitamin C deficit can result in either delayed healing or fracture nonunion.1 It also has other functions in the formation of non- collagenous proteins during bone healing,4 as well as an important role in the development of mesenchymal,5 chondroblast6 and osteogemc7,8 phenotypes.

Vitamin C is also an important reducer. Because a fracture provokes inflammation and the generation of free radicals, reducing substances such as vitamin C may be beneficial in neutralising these harmful elements.9 Although some authors have reported beneficial effects,9,10 others warn that in the presence of metals such as iron, a reducing substance such as vitamin C could generate more free radicals via the Fenton reaction.11,12

Vitamin C deficits are rare in developed countries, except in certain groups such as the elderly,13-15 alcoholics16 and smokers,16 whose vitamin C levels are lower than in the general population (subclinical deficit). A severe vitamin C deficit can seriously impair fracture healing, but the effect that subclinical hypovitaminosis C has on bone healing is unknown.

The aim of this study was to determine whether a subclinical vitamin C deficit interferes with fracture healing in an animal model, and whether healing returns to normal when supplementary vitamin C is administered.

Materials and Methods

The study followed the ethical requirements of the American Physiology Academy17 and was accepted and supervised by the Committee on Research and Animal Care of our centre.

We studied 80 female Osteogenic Disorder Shionogi (ODS) rats (Clea Inc., Tokyo, Japan) aged between 12 and 13 months. These rats have a mutation in the gene coding for the production of the enzyme L-gulonogammalactone oxidase (similar to the enzyme in humans), meaning that they cannot produce their own vitamin C; they are thus suitable for the study of vitamin C deficiency.18

The rats were housed at 22C with light/ dark cycles of 12 hours. They were given unrestricted access to water and a standard vitamin C-free diet. The animals were divided at random into two groups of 40. The control group received 1 mg/ml^sup 4^ vitamin C (Industrias Farma-Quimica Sur, Malaga, Spain) in their drinking water, and the vitamin C-deficient group received 0.5 mg/ml.

We identified the subclinical dosage in the vitamin C-deficient group in a pilot study in which ten ODS rats were randomly divided into five groups of two rats each, and were given dosages of 0, 0.125, 0.25, 0.5 and 1.0 mg/ml vitamin C, respectively. After seven weeks, all rats in the 0 mg/ml group had died, and those in the 0.125 mg/ml and 0.25 mg/m) groups showed signs of scurvy such as spontaneous bleeding and hair loss. Those in the 0.5 mg/ml and 1.0 mg/ml groups showed no symptoms of deficit, and so in the present study the dosage 0.5 mg/ml was used as the subclinical deficiency dosage.

Fig. 1

Radiograph showing a typical transverse fracture of the femur with a Kirschner wire inserted.

An experimental bilateral fracture was made in the femora of all the rats.19 An apparatus was constructed using the principles established by Bonnarens and Einhorn.19 The rats were anaesthetised with an intraperitoneal injection of a solution composing 25 mg/ml of ketamine chlorhydrate (Ketolat, Parke Davis Laboratories, Barcelona, Spain), 0.1 mg/ml atropine (Atropina Braun, Braun Laboratories, Barcelona, Spain), and 0.4 mg/ml diazepam {Valium, Roche Farma Laboratories, Madrid, Spain), administered at 0.25 ml/ 100 mg of the animal’s weight. After anaesthesia, an antibiotic (cephazoline; Kefol, Irisfarma, Madrid, Spain) was administered at a dosage of 4 mg/100 mg of the animal’s weight into the right brachial triceps. To minimise post-operative pain after the fracture, all the rats received 0.10 mg/100 mg weight of buprenorphine 0.3 mg/ml (Buprex, Shering-Plough Laboratories, Madrid, Spain) by subcutaneous injection. Before the fracture was made an intramedullary Kirschner wire 1 mm in diameter was inserted into the right femur. For this, an anterior approach was made to the knee, lateralising the patella and exposing the two femoral condyles. The wire was inserted through the intercondylar line without drilling. The incision was sutured with 3/0 silk and 3/0 absorbable sutures.

The fracture was made immediately afterwards and confirmed by a dental X-ray machine (dental Trophy 70 CCX, Iri Paris, France: exposure 0.10 s, 70 Kv, 80 mA) (Fig, 1 ), When no fracture resulted, the process was repeated, and when the fracture was not transverse the animal was removed from the experiment. After the procedure, the rats were placed in their cages, and the affected limbs were allowed to bear weight a few days later,

After the fracture was made, the two groups were again divided randomly, into four groups of 20 rats each. Two of the subgroups were maintained with the original dosage: controls with 1 mg/ml vitamin C, and vitamin C-deficient with 0.5 mg/ml. The other two subgroups received supplements: control-supplemented with 2 mg/ml (a randomly high dosage, twice the normal dose), and deficitsupplemented with 1 mg/ml (to normalise the rats with deficits).

Five weeks after the fracture, the rats were killed.19 The femora were removed, cleaned of soft tissue, and the intramedullary wires were taken out. None of the wires presented angulations of more than 5. The bones were wrapped in gauze soaked Jn physiological salt solution and preserved at -80C until tested.

Blood levels of vitamin C were measured during the experiment. Just before the rats were killed, blood was extracted from the jugular vein according to the method of Waynforth,2″ The level of vitamin C was determined by high-performance liquid chromatography using the method of Koshiishi and Imanari.21

The right femora were submitted to a low-speed torsion test ( 107min) using a SERVOSIS machine, model MT-IO + PCD-2k; Servosis Enterprise Pinto, Madrid, Spain), with a sensor capable of detecting pressures of less than 10 mNm. Adhesive cement (Osteopak, Biomet- Merck, Sjobo, Sweden) was applied to the ends of the bones to 6t them into the heads of the machine and avoid slippage. Each test provided the following information: XY curve (torque on the bone), maximum torque applied before breakage (maximum torque), angle deformation by maximum torque, sample rigidity calculated by the slope of the XY curve (between 10% and 50%), and energy absorbed by the sample, calculated as the area below the XY curve.

The histological test was performed with the left femora, After thawing at room temperature, the bone was fixed with buffered formol (pH 7.4) for 24 hours and decalcified with nitric acid-formalin for 24 hours. The tissue was then embedded in paraffin and sliced using a high-precision rotation Minot microtome (Leica Microsistemas SA, Barcelona, Spain), providing a series of single, thin sections (3 m; in the sagittal plane). The sections were placed over the microscope slides, removing the paraffin and hydrating them for staining with haematoxylin and eosin. The central section of each piece was used. The samples were digitised with a digital camera (Nikon Coolpix 4500, Barcelona, Spain) equipped with an optical microscope and a specific visor in which the areas of the different zones in each section were delimited. The different areas of the tissues that formed the fracture callus (bone, cartilage, fibre) were measured by the University of Texas Health Science Center at San Antonio image tool program. An area of every tissue was Jn the section. Next, a single blinded observer (TAM) examined the data and the stained slides. The fracture calluses were graded according to the amount of fibrous tissue, cartilage, woven bone and mature bone.22,21 Grade 1 indicated fibrous tissue, grade 2 predominantly fibrous tissue with some cartilage, grade 3 equal amounts of fibrous tissue and cartilage, grade 4 all cartilage, grade 5 predominantly cartilage with some woven bone, grade 6 equal amounts of cartilage and woven bone; grade 7 predominantly woven bone with some cartilage, grade 8 entirely woven bone, grade 9 woven bone and some mature bone, and grade 10 lamellar (mature) bone.

Table I. Mean (\SD) blood levels of vitamin C at death and comparison of levels between the groups

Fig. 2

Graph showing the blood levels of vitamin C at death and the maximum torque of the femur.

The quantitative data were expressed as mean and SD. Normality was confirmed by the Kolmogorov-Smirnov test (SPSS statistical software, Chicago, Illinois). The groups were compared with two-way analysis of variance (ANOVA). The significance level was set at p = 0.05 with the 95% confidence interval (CI) for the means being compared. In order to compare the quantities of vitamin C in the different groups of rats, an ANOVA test for multiple comparisons was performed. Pearson’s correlation coefficient was also used to determine the effect of vitamin C consumption on callus strength in the healed femur before breakage.

Results

Of the 80 initial rats, 18 died in the immediate postoperative period (< 24 hours) and four in the late post-operative period (> 24 hours). No animal was removed from the experiment because all fractures produced were transverse. In two cases the fracture process was repeated, as no fracture had been produced at the first attempt. There were 58 rats remaining by the end of the study, 16 in the vitamin C-deficient group, 11 in the deficient-supplemented group, 15 in the control group and 16 in the control-supplemented group.

The vitamin C blood levels at death varied according to the group. The vitamin C concentrations in rats before death are presented in Table I. A total of 12 samples were discarded because of errors when handling samples.

Figure 2 shows the positive correlation between final vitamin C values and the maximum torque values of the femora. This was significant (p < 0.05), with a correlation coefficient of r = 0.525.

The mechanical strength of the healing femora was higher in the groups with a higher intake of vitamin C. The means (SD) for torque, angle, rigidity and absorbed energy for the mechanical test for the fracture calluses are shown in Table II. The maximum torque values, which are the most critical measure of strength, are shown in Figure 3. All femora broke through the callus (White biomechanical stages I to II}.24

The histological grades of the different tissues indicated slower healing, defined as a lower quantity of bone tissue and cartilage together with a higher quantity of fibrous tissue, in the vitamin-C- deficiem group. The results of the histological tests are given in Table III.

All statistical comparisons between the groups are shown in Table IV.

Discussion

The results of our study indicate that bone healing was enhanced in rats with higher blood levels of vitamin C. The mechanical resistance of the fracture callus and the histological grade of the callus were higher in supplemented groups than in the deficient groups. Furthermore, there was a high correlation between the blood vitamin C levels at death and the mechanical resistance of the calluses formed.

Table II. Mechanical test results, presented as mean (SD)

Fig. 3

Maximum torque (mean and SD) In each group ID, vitamin-C deficient; DS, deficient-supplemented; C, control; CS, control- supplemented).

Table III. Mean (SD) histologtcal grade22,13 (see text) in each group

The effect of vitamin C on fracture healing was studied experimentally Jn rats because non-invasive methods of determining the mechanical strength of the fractured callus are not yet sufficiently developed Jn humans.25 The simplest and most reproducible fracture model appears to be that of Bonnarens and Einhorn,19 and the best way to evaluate the healing is through mechanical testing.25 Of the four parameters that can be measured in the mechanical test (maximum torque, angle, rigidity and absorbed energy), the most important is maximum torque, because it indicates the maximum shear force resisted by the callus before it breaks.19

Standard rats are not suitable for this study because they are able to produce their own vitamin C.18 Humans are unable to produce their own vitamin C, so that vitamin C blood levels solely reflect oral intake. The ODS rat is also unable to produce vitamin C, and thereby provides a good model in which to investigate changes in bone formation in a vitamin C-deficient animal.18 It has been shown that there is an impairment of fracture healing in ODS rats that is completely reversed when they are fed with 1 gfl of vitamin C in drinking water.4

The blood levels of vitamin C were measured before the rats were killed in order to determine whether the groups were appropriate. All the measurements fell within the expected range (Table I), but the expected differences were not found when comparing the vitamin C- deficient and deficient-supplemented groups or the vitamin C- deficient and control groups, probably because of the small sample size. The sample was small because of various factors including the high cost of the animals in Europe (they were imported from Japan), which limited each group to 20 animals, the high post-operative mortality, the difficulty of extracting the serum, and the demands of the high-performance liquid chromatography test to determine the exact vitamin C levels, which caused some samples to be discarded. Despite this, the groups were sufficiently large to yield significant results and, therefore, the results of the mechanical/ histological tests can be ascribed to the different quantities of vitamin C.

Of 80 rats in the study, 18 died in the immediate postoperative period, most likely because of their advanced age. The mean lifespan of these rats is unknown, but it is thought to be shorter than that of Wistar rats,4 estimated to be 18 months.25 Moreover, rats from all four experimental groups died, indicating that death was not a result of vitamin C deficit.

The mean maximum torque in the vitamin-C-deficient group was lower than in the control group (p = 0.001) (Fig. 3). Although statistically significant differences were found between these two groups, when the supplements were provided after the fracture (deficient-supplemented vs control) no difference was shown. This finding was confirmed by the histological test. These results indicate that a subclinical deficit of vitamin C impairs fracture healing in elderly ODS rats, an effect that did not appear when vitamin C was supplied during the healing period. Moreover, supplementation of vitamin C after fracture in the subclinical deficit group (vitamin C-deficient vs deficient-supplemented group) enhanced the histological grade (p = 0.0043) and the torsional strength, but there was no statistical difference, probably because of the small sample size.

Table IV. Statistical comparisons between the groups

It is known that clinical vitamin C deficiency (scurvy) can delay bone healing,2,3 but this study confirms that subclinical vitamin C deficiency can also delay fracture healing. More studies are needed to extend these results to humans. Given the high level of subclinical vitamin C deficit in the elderly population in developed countries,13-16 it might be helpful to prescribe dietary vitamin C supplements during bone healing.

When the control and control-supplemented groups were compared, significant differences were found in the mechanical resistance of the calluses (p = 0.044), with supplemented rats having greater resistance. However, the histological study showed no significant difference between the group supplemented above the normal dosage (controlsupplemented) and those given the normal dosage (control, and deficient-supplemented).

The results of the mechanical test are in agreement with previous findings9,10 that vitamin C supplementation had positive effects on bone healing. However, those studies evaluated only the histological results. In our study we included a mechanical test, which is a rigorous way of evaluating fracture healing.25

If these results are found to apply to humans, the administration of vitamin C supplements during bone healing in non-deficient patients would not simply have no negative effect, but would actually improve bone healing.

There was a significant correlation (p < 0.05, r = 0.525) between the blood level of vitamin C at sacrifice and the mechanical resistance of the fracture callus (Fig. 2). Therefore, the mechanical test indicated rhat the greater the level of vitamin C, the greater the torque resistance of the callus. This finding is in agreement with work by others.9,10

The theoretical harmful effect of the presence of iron (stainless steel from the intramedullary wire) and vitamin C (Fenton reaction) on fracture healing, proposed by Halliwell et al,11 and Halliwell and Gutteridge12 was not shown in this study.

In conclusion, a subclinical deficiency of vitamin C adversely affects fracture healing in elderly ODS rats, although this can be overcome when vitamin C supplements are given during the healing period. The supply of dosages higher than normal during healing enhances the mechanical resistance of the callus.

If these results are found to be similar in humans, the administration of vitamin C during fracture healing may be indicated in the elderly.

The authors would like to thank the staff of the University of Jan Laboratory and the Spanish government, which funded this study through grant FIS 1045/ 01 (Fondo de Investigation Sanitaria).

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

References

1. Phillipt CL, Veowell HN. Vitamin C, collagen biosynthesis, and aging. In: Packer L1 Fuchs J. eds Vitamin C in health and disease New York, USA: Marcel Dekkef Inc., 1997205-30

2. Day SM, Ostnim RF, Chao EYS, el al. Bone injury, regeneration, and repair. In: Simon SR, ed. Orthopaedic Basic Science. 2nd ed. Rosemont, Illinois: MOS. 2000:371-401

3. Michelton J, Cohtn A. Incidence and treatment of fractures in Thalassemia. J Orthop Trauma 1988:2:29-32.

4. Sugimoto M, Hirata S, Sato M, et al. Impa\ired expression of noncollagenous bone matrix protein mRNAs during fracture healing in ascorbic acid-deficient rats. J Bone Miner Res 1998;13:27l-8

5. Francnchi RT. The role of ascorbic acid in mesenchymal differentiation. Nutrition Reviews 1992:50:65-70.

6. Sullivan TA, Uschmann B, Hough R, Leboy PS. Ascorbale modulation of chondrocvte gene expression is independent of its role in collagen secretion J Biol Chem 1994;26922500-6

7. Frenceichi RT, lyer BS. Relationship between collagen synthesis and expression of theosteoblastphenotypeinMC3T3-E1 cells. J Bone Miner Res 1992.7:235-46.

8. Harada S, Matsumoto T, Ogata E. Role of ascorbic acid in the regulation of proliferation in nsteoblast-like MC3T3-E1 cells. J Bone Miner Res 1991;6:903-6.

9. Sarisfizen B, Durak K, Dincer G, Bilgtn OF. The effects of vitamins E and C on fracture healing in rats J lnt MedRes 2002:30:309-13

10. Yllmalz C, Erdemli E, Selek H, M el. The contribution of vitamin C to healing of experimental fractures Arch Qrthap Trauma Surg2001:121:426-8

11. Halliwell B. Gutteridge JMC, Croft CE. Free radicals, antioxidants, and human disease where are we now? J Lab Clin Med 1992:119 59B-620.

12. Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids Arch Biochem Biophys 1990,280.1-8.

13. Richardson TI, Ball L, RoMnfild I Will an orange a day keep the doctor away? Postgrad Med J 2002:78:292-4.

14. Oaffingtr KC. Scurvy: more than historical relevance. Am Fam Physician 1993:48:609-13.

15 Falch JA, Mowe M, Bohnur T, Law levels of serum ascorbic acid m elderly patients with hip fraclure. Scand J CIm Lab Invest 1998:58:225-8

16. Jrvinen R, Knekt P. Vitamin C, smoking and alcohol consumption. In: Packer L Fuchs J, eds. Vitamin C in health and disease. New York: Marcel Dekker Inc. 1997:425-57.

17. Institute for Laboratory Animal Research (ILAR). http:// www.nap.edu/readingroom/books/labfats {date lasl accessed 15 July 2006).

18. Tsunenari T, Fukase M, Fujita T. Bone histormorphometric analysis for the cause of osteopenia in vitamin C-deficient rat (ODS rat). Catcif tissue lnt 1991 ;48 1B-27.

19. Bonnarent F, Einhorn TA. Production of a standatd closed fracture in laboratory animal bone J Orthop Res 1984;2:97-101.

20. Waynforth HB. Methods of obtaining body fluids. In: Waynforth HB, Flecknell PA, eds Experimental and surgical technique in (tie rat 2nd ed. London; Academic Press. 200168-99

21. Koshiishi I, lmanari T. Measurement of ascorbate and dehydroascorbate contents in biological fluids AnalChem 1997:69:216- 20.

22. Haleem AA, Rouie MS, Lewallen DG, el al. Gentamicm and Vancomicin do not impair experimental fracture healing Ctm Orthop2004:427:22-4.

23. Altaian R, Latla L Keer R, at el. Effect of nonstaroidal anliinflammatory drugs on fracture healing a laboratory study in rats. JOrthop Trauma 1995:9:392-40X1.

24. White AA 3rd, Panjabi MM, Southwiek WO. The four biomechanical stages of fracture repair J Bone Joint Surg [Am] 1977;59-A:88-92.

25. Delgado-Martinez AD, Martinet ME, Carraical MT, Rodriguei- Avial M, Munmra L tflecl ol 25-OH vitamin O on fracture healing in eldeily rats. J Orthop Res 1998:16 650-3.

T. AlcantaraMartos,

A. D. Delgado-Martinez,

M. V. Vega,

M. T. Carrascal,

L. MunueraMartinez

From Universidad de

Jan, Jan, Spain

* T. Alcantara-Martofl, MD,

PhD. Orthopaedic Surgeon

Hospital “San Agustin”, Avda

de San Cristobal SIN. 23700

Linerea, Jan, Spain.

* A. D. Delgado-Martinez, MD, PhD, FEBOT, Orthopaedic Surgeon, Professor

* M. V. Vega, MD. PhD,

Orthopaedic Surgeon Assistant

Department of Surgery

Ed. B 3, Universidad de Jen,

Campus Lagunillas S/N, 23071

Jan, Spain.

* M.T. Carrascal, PhD,

Industriel Engineer, Professor

ETS Ingenleros Industriales,

UNED, Apdo Correos 60149,

2B080, Madrid, Spain.

* L Munuera-Martinez. MD,

PhD, Orthopaedic Surgeon,

Professor

Department of Surgery

Universided Autonome de

Madrid, C/Arzobispo Morcillo

S/N. 28046 Madrid. Spain.

Correspondence should be sent to DrT. Alcantara-Martos at San Sebastien. 75 23840 Torredelcampo, Jan, Spain; e-mail: toalma@telefonica.net

2007 British Editorial Society of Bone and Joint Surgery

doi:10.1302/0301-620X.89B3. 16007 $2.00

J Bone Joint Surg [Br] 2007;89-B:402-7.

Received W April 2006; Accepted after revision 1 November 2008

Copyright British Editorial Society of Bone & Joint Surgery Mar 2007

(c) 2007 Journal of Bone and Joint Surgery; British volume. Provided by ProQuest Information and Learning. All rights Reserved.