Growth potential of different zones of the growth plate—an experimental study in rabbits

Growth arrest is one of the major complications after injury of the growth plate in children leading to limb deformity and length discrepancy. The incidence of significant growth disturbance after physeal injuries in children is reported to be between 1% and 10%.1, 2

Surgical manipulation of the growth plate for fixation of physeal fractures can lead to partial or total growth arrest. Despite the intensive research the exact mechanisms that lead to growth arrest are still unknown. Various numbers of different surgical procedures have been described for the treatment of growth disturbances in children. Transposition osteotomy and epiphysiodesis are used for the correction of post-traumatic growth disturbances such as difference in length and axis deviation due to epiphyseal–metaphyseal osseous bridging.3, 4 Newer concepts to improve the causal mechanisms such as loss of epiphyseal cartilaginous cells and the impaired vascularization are still in an experimental status. Special efforts focus on the resection of the osseous bridges and to fill the defect with special materials (e.g. fat, bone wax, apophyseal cartilage, etc.) to prevent osseous bridging. Osterman5 successfully used heterologous rib cartilage as placeholder in epiphyseal defects. In another experimental study ulnar defects in rabbit were filled with autologous chondrocytes cultured from the auricular cartilage with satisfactory results.6 Subsequently, this method was used in eight children. The author reported that the resection of the osseous bridges with consequent transplantation of chondrocytes rebuilt functional epiphyseal cartilage.6 Rudolph et al.7, 8 compared the use of autologous epiphyseal cartilage to that of frozen homologous epiphyseal cartilage in rabbits to prevent epiphysiodesis. They demonstrated that the autologous but not the homologous cartilage transplant was able to prevent osseous bridging. However, despite the clinical efforts to treat growth disturbances only little is known about the growth potential of the different zones of the growth plate. For example, there is little known about which parts of the growth plate can be cut for transposition osteotomy or which part of the growth plate can be safely fixed by implants.

Therefore, the aim of this study was to investigate the growth potential of different zones of the growth plate. We further aimed to investigate the effects of the destruction of the epiphyseal and metaphyseal vascular system through osteotomy.

MATERIALS AND METHODS

Surgical Procedure

The animals were deprived of food for 24 h and received atropine-sulfate as premedication. The anesthesia was induced and maintained by ketamine (35 mg/kg, i.m.) and xylacin (5 mg/kg, i.m.). After positioning of the animals in the prone position both forelegs were shaved and prepared aseptically for the operation. A dorso-lateral incision of the distal ulna between the extensor digitorum and extensor carpi ulnaris muscle was made.

Group I (n = 12) En-Bloc Resection of the Proximal (Metaphyseal) Segment of the Growth Plate

Resection of the growth plate cartilage was performed between the middle and distal third of the growth plate. This area has been reported to be the border between reserve and proliferation zone.9 An osteotomy was performed between the reserve and proliferation zone using a surgical chisel. A second osteotomy was performed 25 mm proximal from the first osteotomy at the diaphysis. The bloc between both osteotomies was then removed (Fig. 1).

Group II (n = 6) En-Bloc Resection of the Distal Segment of the Growth Plate

An osteotomy was performed between the reserve and proliferation zone using the surgical chisel. A second osteotomy was performed at a distance of 4 mm distal from the first osteotomy. The bloc between both osteotomies was then removed (reserve zone) (Fig. 1).

Group III (n = 6) En-Bloc Resection of the Complete Growth Plate and Re-Implantation

The complete growth plate was removed after two osteotomies (epiphyseal and metaphyseal osteotomies parallel to growth plate). Thereafter, it was cleaned from diaphyseal and epiphyseal tissue parts, to interrupt epi- and metaphyseal vascular supply, and again re-implanted within the created defect (Fig. 2).

Group IV (n = 6) Resection of the Growth Plate and Re-Implantation After 180° Rotation

After resection of the complete growth plate and removal of meta-/epiphyseal parts as described above, it was rotated and re-implanted in an upside down position (Fig. 2).

Group V (n = 10) Control Group

No surgical intervention was performed in this group.

Animals were allowed to recover for 6 h and fed at standard maintenance diet and provided water ad libidum. All animals received suppository with 0.3 g natrium-phenyl-dimethyl-pyrazolonum-methylamino-methynsulfonicum as analgesic therapy.

RESULTS

Group I

The en-bloc resection of epiphyseal cartilage adjacent to the metaphysis which was removed together with a piece of the diaphysis of about 25 mm thickness at the left ulna, did not result in any bone deformation. Minor growth retardation of the bone was observed which was compensated after 2–4 weeks by an enhanced enchondral ossification. Tissue sections contained a strongly thickened epiphyseal plate with chondrous columns of 60–90 cells. The hypertrophic zone, however, did not show typical signs of remodeling with edema, vacuoles and beginning calcification (Fig. 3). Radiologic examination after 2 and 12 weeks showed a progressive correction of the created defect (Fig. 4).

Group IV

En-bloc resection of the growth plate and re-implantation after 180° rotation caused a temporary growth delay which completely recovered 12 weeks after surgery (Fig. 5). The histological examination showed vascular connection of the growth plate to the epi- and metaphyseal vascular system already after 2 weeks. A complete reintegration of the rotated growth plate without any signs of premature ossification was observed after 12 weeks (Fig. 6).

DISCUSSION

This study demonstrates that en-bloc resection of the proximal two-thirds of the epiphyseal plate with a metaphyseal segment and the adjacent diaphyseal segment at the border between the reserve zone and the proliferation zone does not influence the osseous growth. The resection corresponds to a type-I Salter and Harris lesion. The growth delay following trauma is compensated after 2–4 weeks. However, as a reaction to the disruption of the metaphyseal blood supply a considerable broadening of the cartilage was observed. In the tissue sections hypertrophic cartilaginous cells at both ends of a column were seen. These cells were similar in size as the cartilaginous column cells of a normal epiphyseal plate. The only morphologic difference to the normal growth plate was that in an undamaged epiphyseal plate the proliferative cartilaginous cells enlarge and become hypertrophic only in the middle of the column. Though these alterations after injury were transient and did not influence the normal osseous growth. These results are in line with the results reported by von Laer10-12 who suggested that epiphysiolysis are considered as diaphyseal fracture and therefore should be treated conservatively. We therefore believe that the metaphyseal segment of the growth plate is not very important for the growth process.

Total removal of the epiphyseal part of the growth plate led to the resorption of the cartilage within 2 weeks resulting in axis deviation and growth retardation of the bone. Perichondral repair mechanisms as described by different authors could not compensate the tissue damage.9, 13-16 These results confirm previously reported results of our group demonstrating that drilling and screw fixation of the growth plate through the epiphyseal side of the growth plate lead to severe growth disturbances in rabbit ulna.17 Whereas, and screw fixation of the growth plate through the metaphyseal side of the growth plate only led to temporary growth disturbance which was compensated within a short time. These results demonstrate the importance of the epiphyseal (reserve) zone of the growth plate for the growth process. Hence it follows that the reserve zone has the highest growth potential of the entire growth plate.

Another aim of this study was to investigate the histological and radiological consequences of fracture or the osteotomy of the growth plate for the osseous growth. Transposition osteotomy and epiphysiodesis are used for the correction of post-traumatic growth disturbances such as difference in length and axis deviation due to epiphyseal–metaphyseal osseous bridging.3, 4 Newer concepts to improve the causal mechanisms such as loss of epiphyseal cartilaginous cells and the impaired vascularization are still in an experimental status. Special efforts focus on the resection of the osseous bridges and to fill the defect with special material (e.g., fat, bone wax, apophyseal cartilage, etc.) to inhibit osseous bridging. As previously mentioned correction of post-traumatic growth disturbance often requires osteotomy and epiphysiodesis.1-22 Newer concepts for the growth disturbance are in an experimental status. A Chinese group described in a well-conducted study the transplantation of autologous growth plate chondrocyte from iliac crest epiphyseal cartilage into the tibial growth plate defect of rabbits.20 Their results demonstrated that the treatment of growth plate defects with tissue-engineered composite established by combination of autologous growth plate chondrocytes and DBM can prevent the formation of a bone bridge and restore the growth of damaged growth plate. In another recent experimental study Coleman et al. used agarose hydrogel to treat growth plate injuries in rats. Their results showed that the treatment of growth plate injuries with agarose hydrogel reduced limb length discrepancy but was not sufficient to regenerate growth plate tissue or fully restore growth function.19

Another experimental treatment option is the use of mesenchymal stem cells. Autologous bone marrow MSC were transplanted into a surgically created defect of the proximal ovine tibial growth plate. However, the examination of implants at 5-week post-operatively revealed that transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Moreover, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated.21

Other studies deal with the cellular mechanisms of the growth disturbance after injury of the growth plate. Chung et al. analyzed in a recently published study the role of PDGF-BB in the bony repair of the injured growth plate. The demonstrated that the inhibition of PDGF-BB in rats with growth plate injury lead to exhibition of less bony trabeculae and osteoclasts at the injury site. Their results suggested that PDGFBB contributes to growth plate injury repair by promoting mesenchymal progenitor cell infiltration, the chondrogenic and osteogenic responses, and remodeling of the repair tissues.18 Another study showed the involvement of the bone morphogenic proteins (BMP 2, 3, 4, and 7) in the fibrogeneic, osteogenic, and remodeling responses at the injured growth plate of young rats.22 Considering these results it would hypothetically be conceivable to inhibit the mentioned BMPs or PDGF-BB to prevent osseous bridging of the growth plate.

All mentioned techniques require osteotomy within or around the growth plate. The osteotomy, however, leads to destruction of the epi- and metaphyseal vascular system of the growth plate. It is conceivable that the destruction of the vascular blood supply leads to destruction of the growth plate. In this study en-bloc resection of the whole epiphyseal cartilage was performed to simulate meta- and epiphyseal osteotomies and to disrupt the vascular blood supply. The epiphyseal cartilage was then re-implanted. We observed a transient growth retardation that was compensated after 12 weeks. We therefore suggest that the growth disturbance must be based on cellular but not on vascular diminution. In summary the results of this study showed that the reserve zone is essential for the functioning of the growth plate. Injuries of the reserve zone result in axis deviation and growth retardation of the bone. Growth disturbances are based on a cellular level. Transplanted growth plates with an intact reserve zone can find vascular connection and continue to growth.