Session I - Basic Science

Anabolic Enteral Protein Supplementation Enhances Soft Tissue and Fracture Healing in a Malnourished Rat Model

Timothy A. Burd, MD; Jeffrey O. Anglen, MD; James L. Cook, PhD; Peter Kazmier, MD; Michael S. Hughes, BS, University of Missouri, Columbia, Missouri, USA (OTA Grant)

Purpose: We examined the effects of protein malnutrition and subsequent anabolic amino acid supplementation on skeletal muscle and fracture healing.

Methods: We studied 100 adult male Sprague-Dawley rats; 10 animals served as controls, receiving a 15% protein diet throughout the study. The remaining 90 rats received a 6% protein diet for 5 weeks to induce a state of protein malnutrition. Serial venous samples were obtained from the rats' tail vein to assess total lymphocyte count and serum albumin, both well-accepted serologic markers of overall malnutrition. After 5 weeks, three groups were formed (30 rats/group). Group A received a 6% protein diet, group B received a 15% protein diet, and group C received a 30% anabolic protein diet, of which 50% was composed of the conditionally essential amino acids glutamine, arginine, and taurine. All three diets were isocaloric and identical in content except for the 30% percent protein group. After the 5-week malnourishment phase, all 90 animals underwent femoral intramedullary nailing with 0.45-inch Kirschner wire. A retrograde approach through the intercondylar femoral notch was used, followed by a closed, reproducible midshaft femur fracture. At 2, 4, and 6 weeks, 10 animals from each group were sacrificed. The quadriceps muscles and femurs were harvested bilaterally. Serum markers for malnutrition were assessed throughout the study. Dual-energy X-ray absorptiometry (DEXA) and biomechanical testing in torsion were employed to determine differences among the groups.

Results: Malnutrition. Malnutrition was achieved with a 41% decline (3.7 g/dl to 2.18 g/dl) in serum albumin by 5 weeks (P < 0.0001). There were no surgical complications regarding rod placement technique, and more than 90% of all animals had closed, transverse, midshaft fractures created with the remainder usually within the distal one-third of the femoral shaft. All animals ingested a similar amount (~22 grams/day) of food throughout the study, suggesting an autoregulatory phenomenon. That is, the 30% group ate twice as much protein as the 15% group and 5 times the amount compared with the 6% group. Despite isocaloric intake during this period, the 90 malnourished animals weighed 7% less than the 10 control rats on a 15% protein diet (366 grams vs. 391 grams). By 4 weeks after femoral nailing, the anabolic group overall body weight was equivocal to that of the control animals (~425 grams), whereas the 6% (371 grams; P = 0.0001) and 15% (409 grams; P = 0.0019) groups lagged in convalescent weight gain.

Muscle. At 6 weeks after intramedullary nailing, rectus femoris weights were significantly greater in the 30% anabolic diet group compared with the 15% (P = 0.012) and 6% (P = 0.00007) groups. Additionally, the noninjured thigh in the anabolic diet group had greater muscle mass compared with the control, nontraumatized rats (P = 0.014) as well as the 15% protein diet (P = 0.0043) and 6% protein diet (P = 0.0043) groups. There was also a significant difference between the rodded and non-rodded thighs in that the anabolic group demonstrated no difference in muscle mass when the injured and uninjured limbs were compared (P = 0.21). In contrast, the rodded limb in both the 6% and 15% protein groups revealed much less mass compared with their contralateral thighs (P = 0.04 and P = 0.012 respectively).

DEXA. With DEXA analysis, fracture callus bone mineral density was significantly greater (17%) at 6 weeks in the anabolic group (P = 0.0033) compared with the control animals; this phenomenon was not significantly appreciated in the other treatment groups. Bone mineral area, content, and density were greater at 6 weeks in the 30% group when the entire injured femur was compared with that of the control, uninjured group (P < 0.032). Again, this trend was not identified within the 6% or 15% diet animals.

Biomechanics. The strength of healing callus and the contralateral, noninjured femur were tested in torsion to ultimate failure. Only the anabolic group demonstrated a significant increase in callus strength from 2 to 4 weeks testing (13.5 N vs. 27.4 N, P = 0.013). The 6% and 15% protein groups steadily increased in fracture strength such that there were no distinct differences among the groups at 6 weeks.

Conclusions: We effectively demonstrated that a malnourished animal model could be achieved with a protein poor isocaloric diet. A novel blunt guillotine apparatus was also created to consistently reproduce a closed midshaft femur fracture in rats. Skeletal muscle mass and overall animal weights increased within the 30% protein group. Both DEXA and biomechanical testing demonstrated increased bone mineral density and torsional strength in the anabolic group. These are encouraging results and suggest that anabolic supplementation by using a high-protein diet along with conditionally essential amino acids may augment fracture callus properties and improve the surrounding peri callus soft tissue muscle mass.

Significance: Identification of pathways to augment skeletal muscle and fracture healing via anabolic oral supplementation may have direct clinical application by mitigating morbidity, specifically nonunion rates, infection, and rehabilitation, as well as mortality in orthopaedic patients.