91
ITSP 6.0
Page 4 of 25
Section C
–
Project Details
1. Background
1.1. General background leading to this project. Please provide supporting documents,
such as published papers by members of the project team or other researchers.
Musculoskeletal disorders are leading cause of disability around the world, accounting for 25% of total
cost of illness
18
. In Hong Kong, 49428 cases (3.2%) of discharges/death related to diseases of the
musculoskeletal system and connective tissue in 2007
4
. The increased recreational sport and elite
consideration have drawn more societal concerns on effective treatment for sport injuries, including various
forms of ligament and tendon injuries, which have taken up significant health care resources. However,
medical research to improve healing outcomes of tendon and ligament injuries is far lagged behind. Our
research team has investigated tendon and ligament healing since 1995. In the past 14 years, we received 9
Research Grant Committee Earmarked grants and 7 CUHK direct grants, generating 88 publications in
peer-reviewed journals. (Annex No.1)
Tendon and ligament injuries are notorious with the slow healing rate and compromised mechanical
restoration. We have studied tendon healing in a rat model of patellar tendon healing up to 6 months post
injury
35
. This rat model mimics the clinical case of patellar tendon injury when the central-one third of the
patellar tendon was used as a graft for ACL reconstruction. In this animal model and in tendon cell culture
studies, we have investigated the effects of growth factors
31,37,17,14,22,27,26,23
, traditional Chinese herbal
medicine
21
and biophysical intervention
10
to promote tendon healing. Recently we also investigated the
beneficial effects of mesenchymal stem cells, biomaterials and bisphophanates (a class of drugs that inhibit
bone resorption) in rabbit
2,11
and rat models of ACL reconstruction (Annex no.2). With these studies, we
established numerous techniques for histological, biochemical and mechanical assessments of tendon and
ligament healing.
In our previous study, we found that cellular activities are changing in different healing stages
15
, and
earlier application of biophysical intervention exhibited better effects to improve healing outcomes
18
. Natural
healing process of tendon and ligament injuries involves four different stages: inflammation, cell recruitment,
granulation and remodeling. Previous attempts to promote tendon and ligament healing mainly targeted on
latter events such as enhancement of cell proliferation and matrix synthesis
23,26,31
; but modulation of earlier
events, especially in the inflammatory stage, has not been explored. Anti-inflammatory treatments are
commonly prescribed to tendon and ligament injuries to relieve symptoms; but its negative effects on tendon
and ligament healing are now clear, since some pro-inflammatory mediators (i.e. prostaglandins) are crucial to
trigger early healing events such as neo-vascularization
3
. It is possible that optimization of these early
healing events during the inflammatory stage of healing will be a more effective way to improve the
healing outcomes. During inflammatory stage of tendon and ligament healing, inflammatory cells are
recruited to the injured sites to tackle with potential microbial infection and clear the damaged tissues. At this
stage, significant oxidative stress prevails and neovascularization occurs. Neovascularization is the most
important event in this early healing stage by bringing healing cells, nutrients and oxygen for the repair
process. Since overwhelming oxidative stress is harmful to tendon cells
20
, reduction of oxidative stress by
antioxidants such as vitamin C (L-ascorbic acid) may exert protective effects. In addition to its powerful
antioxidant activity, vitamin C also exerted protection on vascular cells by modulation of nitric oxide synthase
expression, enhancement of cell growth, attachment and migration in vascular smooth muscles cell
32,13,37
.
Extremely low levels of ascorbate occur in patients and animals after trauma and infection, and early repletion
requires injection of supraphysiological doses of ascorbate
19.28
. These evidence suggest that early
supplementation of vitamin C may have beneficial effects for wound healing. A recent study reported that
intraperitoneal injection of high dose vitamin C promote Achilles tendon healing as revealed by histological
findings
7
. In a chicken model of flexor tendon repair, we found that a single bolus injection of vitamin C after
surgical repair could reduce the extent of tendon adhesion and hence promote functional restoration. It
suggests that local application of vitamin C at very early stages of healing (after surgical repair) can promote
tendon and probably ligament healing, since oxidative stress and neovascularization are important
determinants for both tendon and ligament healing. As Vitamin C has a very high safety profile
24
, it gains the
merits of high applicability for clinical use. Since surgical irrigation saline (sterile isotonic sodium chloride
solution) is used for draining and cleansing during surgical operation, it may be the best carrier for vitamin C
during surgical operation. In the present research project, we propose to develop the use of vitamin C
irrigation solution to promote tendon and ligament healing. pre-clinical trials will be performed in animal
Appendix V ITF proposal on vitamin C irrigation solution
72
ITSP 6.0
Page 5 of 25
models with the advanced assessment methods we have established.
1.2. Any previously related project(s) undertaken by your organization and/or key project
team members in the past five years and supported by the ITF or any other funding
sources in Hong Kong and around the world? If yes, please briefly describe the
relevant/related project(s) and the source(s) of funding obtained for it (them).
In the past 5 years, our project team members have received 4 grants from General Research Fund
(formerly competitive earmarked grant, Research Grant Committee) which are related to the present
proposal (Annex no.1):
1.
Use of Bisphosphonate to Inhibit Local Bone Resorption and Improve Healing of Tendon Graft to Bone
Tunnel. (PI: Prof. Lui PPY, duration: 2009-2011)
In this project, we established a rat model of ACL reconstruction with histological examination on decalcified
samples of the graft incorporation and mechanical test for the ultimate loading of the reconstructed ACL. The
temporal changes of the healing outcomes were recorded. This information will be useful for the present
research project.
2.
Augmentation of Tendon Graft Healing within Bone Tunnel Using In-Situ Setting, Calcium Phosphate
Bone Cement Complement with Autologous Mesenchymal Stem Cells (PI: Prof. Chan KM, duration:
2007-2009)
In this project, we used a rabbit model of ACL reconstruction to investigate the effects of stem cells and
biomaterials. The surgical techniques were modified for the establishment of the rat model of ACL
reconstruction.
3.
The use of antioxidants to reduce tendon adhesion (PI: Prof. Hung LK, duration (2007-2009)
In this project, we evaluated the effect of peritendinous vitamin C/vitamin E injection (immediately after
surgical repair) on the development of tendon adhesion. The results showed that vitamin C given as a single
bolus injection can reduce the extent of tendon adhesion probably thorough its anti-oxidant effects. It inspired
our idea to develop vitamin C irrigation solution to promote tendon and ligament healing in the present
proposal.
4.
An experimental study on the use of low intensity pulsed ultrasound to optimize patellar tendon healing
in a rat model (PI: Prof. LK Hung, duration:2004-2007)
In this project, we used a well-established rat model of patellar tendon injury to evaluate the effect of
therapeutic ultrasound. We found that earlier biophysical intervention by LIPUS exhibited better effects to
improve healing outcomes. The time points of different healing stages in this model (inflammation,
granulation and remodeling) were defined, which will be used as references for sampling time points in the
present proposal.
1.3. Any other R&D work or projects similar to this application done or being carried out
by other parties in Hong Kong and around the world? If yes, please set out the
findings and explain how your approach is compared to others in terms of
technological superiority, production costs, market acceptability, etc.
To the best of our knowledge, we are the first group proposing the addition of Vitamin C in surgical
irrigation solution. From a search in the free patent online (http://www.freepatentsonline.com/), only two
expired and one current items related to surgical irrigation saline was found (US4504493, US4872865,
US20060205657). Modifications of surgical irrigation saline have been proposed in other aspects, including
addition of antiseptics to reduce chance of infection
8
, detergents
25
and supplementation of epinephrine to
reduce bleeding
30,34
. However, up-to-date there are no reports describing the feasibility of modified surgical
irrigation solution to optimize tendon/ligament healing.
A recent study reported intraperitoneal injection of high dose vitamin may promote Achilles tendon
healing as revealed by histological findings
7
. Instead of systemic administration by intraperitoneal injection,
we investigated the effect of peritendinous injection of vitamin C on tendon adhesion in a chicken model of
flexor tendon repair
1
and positive results were reported
6
(Annex no.3). Due to the difficulty to keep injected
vitamin C in peri-tendinous space, we propose to supplement vitamin C in irrigation solution in the present
proposal. This will simplify the application procedure and circumvent the problem of leakage. In a pilot study
we showed that irrigation of 5 ml vitamin C solution significantly increased neovascularization into tendon
wound in the rat model of patellar tendon injury (Annex no.4, 5). It suggested that vitamin C supplementation
may be an added value to surgical irrigation solution for a better healing outcome.
Appendix V ITF proposal on vitamin C irrigation solution
69
ITSP 6.0
Page 6 of 25
With its safety profile
24
, low cost and potential beneficial effects to promote tendon and ligament
injuries, vitamin C irrigation solution would have an advantage in terms of cost, technological feasibility and
market acceptability, as compared to all commercially available surgical irrigation saline. It is foreseeable that
extra intra-operative care will be achieved by this value-added surgical irrigation solution.
1.4. Any pilot work has already done by your organization and/or project team members
in preparation for this project? If yes, please describe the work done.
Our team has established a full range of experimental techniques to evaluate the healing outcomes of tendon
and ligament injuries in rat models:
1.
Animal models
A well-established rat model of patellar tendon injury will be used for evaluation of the effects of vitamin C
irrigation solution on tendon injuries. We have published 12 papers (Annex 1)on this model and monitored the
structural and functional restoration of the injured tendons up to 6 months post injury. The healing outcomes of
patellar tendon injuries are evaluated by histology and mechanical tests as routine. For evaluation on ligament
injuries, we have established a rat model of ACL reconstruction with characterization on histology and
mechanical properties of the graft. A conference abstract based on this model was accepted by International
Symposium of tendon and Ligament X to be held on 5 Feb 2010. (Annex no.2)
2.
Measurement of neovascularization in healing patellar tendon by High resolution 3-D Doppler
ultrasound imaging
We developed the use of high resolution ultrasound imaging to monitor tendon healing in rat patellar tendon
injury (Annex no.6). The changes in wound size, tendon swelling and echo textures of the healing tendon were
monitored up to day 42 post injury. Since the extent of neovascularization is deterministic for healing
outcomes, we also developed a quantitative measurement of vascularity in the tendon wound by 3-D Doppler
ultrasound imaging
12
(Annex no.7). These techniques will be used to evaluate the effects of vitamin C
irrigation solution.
3.
Animal gait analysis to measure painful response associated with tendon and ligament injuries
We have developed the use of video-assisted gait analysis to monitor painful responses in a rat model with
collagenase-induced patellar tendon injuries. We found that the injured rats exhibited abnormal gait
parameters on both the injured limbs and the contralateral control limbs as compared to the control group with
saline injection
5
(Annex no.8). Gait changes associated with pain could also be monitored in rat models of
knee injuries, including ACL deficiency and traumatic patellar tendon injuries. As a result, video-assisted gait
analysis will be employed to evaluate functional changes related to the healing outcomes of injured ACL or
patellar tendons after supplementation of vitamin C in surgical irrigation solution.
4.
Effects of Vitamin C injection on tendon adhesion in a chicken model of flexor tendon repair.
On the potentials of vitamin C supplementation to optimize tendon healing, we have almost completed an
Earmarked grant entitled
“
the use of antioxidants to reduce tendon adhesion
”
. In a chicken model of flexor
tendon repair, we established a new method to evaluate the severity of tendon adhesion (Annex no.10). We
also found that local injection of vitamin C could reduce oxidative stress and reduce tendon adhesion, which
was demonstrated as a decrease in gliding resistance and improved range of motion
6
(Annex no.3). Based on
these findings, we propose that supplementation of vitamin C in surgical irrigation solution may be also be
useful in other tendon and ligament injuries, and a pilot study was performed in a rat model of patellar tendon
injury.
5.
Effects of vitamin C irrigation solution on patellar tendon healing
In a pilot study, we compared the effect of vitamin C irrigation saline (30 mg/mL) on tendon healing with
normal saline as control. During the surgical procedure of induction of patellar tendon injury, either 5 ml of
vitamin C saline or normal saline was used to cleanse the wound. The patellar tendons were washed before
injury was induced, during the removal of central-one-third of the patellar tendon, and after the fascia was
closed by sutures. The preliminary data showed that vitamin C irrigation (n=6) significantly increased
neovascularization in the tendon wound at day 4 post injury as compared to normal saline irrigation saline
group (n=6) (Annex no.4 & 5). It suggests that vitamin C irrigation solution may have a positive effect on the
Appendix V ITF proposal on vitamin C irrigation solution
44
ITSP 6.0
Page 7 of 25
healing outcomes of patellar tendon injury.
1.5. Any request for funding support for this application previously rejected by ITF,
Research Grants Council (RGC) and/or other Government-administered funding
schemes? If yes, please set out the project reference of the previous application.
No, this is a new project.
1.6. If this application is a previously rejected application under ITF, any major differences
of this application vis-à-vis the previous one? Please explain how these differences
have addressed the previously raised concerns.
Not applicable.
1.7. Any attempt made or being made to seek funding support for this project from other
funding sources in Hong Kong and around the world (e.g. RGC, Applied Research
Fund, Environment and Conservation Fund, etc.)? Please advise any known results
of the application(s).
No.
References:
1. Fu SC, Hung LK, Lee YW, Mok TY, Chan KM. (2010) A novel approach to measure tendon adhesion
by video-assisted gliding test in a chicken model. J Hand Surg (Eur). (Accepted)
2. Lui PP, Lee YW, Mok TY, Cheuk YC, Fu SC, Ho G, Chan KM. (2010) The effect of alendronate on the
mechanical strength of the tendon graft to bone tunnel complex after ACL reconstruction. ISL&T X
2010 Hong Kong.
3. Chan KM, Fu SC. (2009) Anti-inflammatory management for tendon injuries – friends or foes? Sports
Med Arthrosc Rehabil Ther Technol. 1:23.
4. Health Facts of Hong Kong 2009 Edition. (2009) Department of Health, Hong Kong SAR.
5. Fu SC, Chan KM, Chan LS, Fong DT, Lui PP. (2009) The use of motion analysis to measure
pain-related behaviour in a rat model of degenerative tendon injuries. J Neuosci Methods.
179(2):309-18.
6. Fu SC, Hung LK, Shum A, Lee YW, Mok TY, Lui PP, Chan KM. (2009) Local vitamin C injection
reduced tendon adhesion in a chicken model of injured flexor digitorum profundus tendon injury.
ISL&T IX 2009 Las Vegas.
7. Ömeroğlu S, Peker T, Türközkan N, Ömeroğlu H. (2009) High-dose vitamin C supplementation
accelerates the Achilles tendon healing in healthy rats. Arch Orthop Trauma Surg 129:281-286.
8. Owens BD, White DW, Wenke JC (2009) Comparison of irrigation solutions and devices in a
contaminated musculoskeletal wound survival model. J Bone Joint Surg Am 91:92-8.
9. Wen CY, Qin L, Lee KM, Chan KM. (2009) Peri-graft bone mass and connectivity as predictors for
the strength of tendon-to-bone attachment after anterior cruciate ligament reconstructions. Bone.
45(3):545-52.
10. Wen CY, Qin L, Lee KM, Chan KM. (2009) The use of brushite calcium phosphate cement for
enhancement of bone-tendon integration in an anterior cruciate ligament reconstruction rabbit model.
J Biomed Mater Res B Appl Biomater. 89B(2):466-74.
11. Wen CY, Qin L, Lee KM, Wong MW, Chan KM. (2009) Influence of bone adaptation on
tendon-to-bone healing in bone tunnel after anterior cruciate ligament reconstruction in a rabbit
Appendix V ITF proposal on vitamin C irrigation solution
Documents you may be interested
Documents you may be interested