Evaluation of the Clinical Effectiveness of Bioactive Glass (S53P4) in the Treatment of Non-unions of the Tibia and Femur: a Randomized Controlled Non-inferiority Trial
Phase: NA
Conditions: Pseudoarthrosis of Bone
Sponsor: Sebastian Findeisen

Efficacy of bioactive glass as a bone substitute in
Masquelet therapy in the treatment of tibial and
femoral pseudarthrosis
Responsible study physician:
Dr. med. Patrick Haubruck, Resident at the Clinic for Orthopedics and Trauma 
Surgery
Study Director: 
Prof Dr. med. Gerhard Schmidmaier 
Deputy Director Orthopedics and Trauma Surgery 
Head of the Trauma Surgery Section 
Others involved in the study: 
Dr. med. Fabian Westhauser, Resident at the Clinic for Orthopedics and Trauma
Surgery
Dr. med. Thomas Ferbert, Resident at the Clinic for Orthopedics and Trauma 
Surgery
Dr. med. Christian Fischer, Senior Physician of the Clinic for Orthopedics and 
Trauma Surgery Dr. med. Michael Tanner, Senior Physician of the Clinic for 
Orthopedics and Trauma Surgery Dipl. rer. soc. Simone Gantz, Biometrische 
Beratung und Projektkoordination Orthopädie und Unfallchirurgie
Sponsor: 
BonAlive Biomaterials Ltd, Biolinja 12, 20750 Turku, Finland 
16.08.2017

1) Summary:
In the treatment of pseudarthrosis, the application of autologous cancellous bone is still
considered the gold standard. However, the possibilities for autologous cancellous bone
harvesting are limited, and the morbidity of the harvesting is a considerable burden for the
patients. Therefore, there is an intensive search for suitable bone substitute materials. 
Bioactive glass (S53P4) is an established synthetic biocompatible bone graft substitute which
stimulates bone formation and improves blood supply in the affected areas. Furthermore,
bioactive glass is established in the therapy of bone inflammation. The therapy of large bone
defects consists of a two- or multi-stage procedure and is called Masquelet therapy. In a first
procedure the bone is locally conditioned and in a second procedure the bone defect is filled
with a combination of autologous bone and tricalcium phosphate. This autologous bone is
obtained from RIA (Reaming-Irrigating- and Aspirating) material of the ipsi- or contralateral
femur or from iliac crest cancellous bone with tricalcium phosphate. However, this requires
another surgical procedure to obtain the bone graft, which is associated with risks and may
cause additional problems for the patient. Multiple studies have shown that filling bone
defects with bioactive glass leads to new bone formation and bone regeneration. This could
lead to the elimination of the need for autologous bone grafting, thus avoiding the need for
further surgical intervention. The planned clinical study will compare the effects on osseus
consolidation of pseudarthrosis at the tibia and femur between standard Masquelet therapy
(G1) and Masquelet therapy using bioactive glass as bone graft substitute (G2). The study
will  include  50  patients  with  pseudarthrosis  of  the  femur  and  tibia  in  a  prospective
randomized design. After randomization, 25 patients will be treated with either Masquelet
therapy (G1) or Masquelet therapy using bioactive glass as replacement material (G2). 
The clinical course will be evaluated preoperatively, 2 days, 6 and 12 weeks and 6,9, 12 and
24 months postoperatively. In addition to clinical and nativradiological standard control
examinations, perfusion is examined by CEUS ultrasound (contrast enhanced ultrasound)
and  dynamic  contrast  enhanced  MRI  (DCE  -MRI)  preoperatively  and  12  weeks
postoperatively. Furthermore, blood samples are taken to check the infection levels, local
blood flow situation and bone growth markers.
2) Introduction / Scientific Basis:
Bone has the ability to regenerate after injury in a complex physiological process. However,
this process can be disturbed in extensive injuries and critical blood supply or infectious
situations and delayed fracture healing is a serious complication in trauma surgery (1).
Depending on the individual patient risk, 5-10% of all fractures lead to pseudarthrosis. The
tibia is most frequently affected (2). Pseudarthrosis is not only a common complication but
also leads to serious consequences. Pain, immobilization, and loss of the affected limb pose
significant limitations for patients (3). The complex pathophysiology of delayed or failed
fracture  healing  requires  multifactorial  treatment.  The  so-called  Diamond  concept  (4)
consists of the following components:
- Biomechanical stability: angular stable plates, dynamic intramedullary nails.
- Osteogenic cells: mesenchymal stem cells, autologous cancellous bone (iliac crest or
RIA)

- Osteoconductive structures: autologous cancellous bone, synthetic bone substitutes
- Growth hormones: bone morphogenic proteins (BMP-2 and BMP-7)
- Circulation: by improving macro- and micro-circulation, local induction of a 
Masquelet membrane and debridement of fracture ends.
Although  autologous  bone  grafting  is  the  gold  standard  for  filling  bone  defects,  it  is
associated with great morbidity for the patient and availability is limited (2). Therefore, in
the last decades, there has been an intensive search for suitable bone graft substitutes that
could lead to new bone formation and reduce the need for autogenous bone grafting in the
future. Initial studies have shown that bioactive glasses have an outstanding antibacterial
effect,  which can  be  attributed to an  increase  in  local  PH  and  osmotic  pressure  (5).
Surprisingly, during follow-up of these initial studies, scientists found extensive new bone
formation in patients treated with bioactive glass. This led to intensive research efforts to
investigate bioactive glass as a bone graft substitute. It was found that bioactive glass with
the composition of SiO2, Na2O, CaO and P2O2 initiates growth of tissue and stimulates bone
growth (5-9). The effect of bioactive glass is based on the release of ions (Si+, Na+, Ca2+) and
their reaction with body fluids. After application in situ, these surface reactions lead to the
formation of a calcium phosphate layer that bonds with solid materials such as bone (10).
Numerous studies have demonstrated the clinical efficacy of this mechanism in oral and
maxillofacial  surgery  (11,  12),  spine  surgery  (13),  and  trauma  surgery  (5,  7-9,  13).
Furthermore,  numerous  studies  demonstrated  the  safety  of  the  clinical  application  of
bioactive glass for filling bone defects (5, 7-9, 13). However, there are no clinical studies
evaluating the use of bioactive glass in masquelet therapy of pseudarthrosis. Contrast-
enhanced ultrasound (CEUS) as well as dynamic contrast-enhanced magnetic resonance
imaging can visualize the vascularization of pseudarthrosis in real time. In this regard, CEUS
and  DCE-MRI  can  be  used  preoperatively  to  differentiate  between  infectious  and
noninfectious pseudarthroses (14, 15). Postoperatively, DCE- MRI provides a valid tool for
predicting bony consolidation of pseudarthrosis (16). Perioperative cytokine measurement
of bone growth markers is a well-established method that provides an early indication of
whether local infection is present or healing is likely (17, 18). 
Bioactive glass (S53P4) is approved for the treatment of bone defects in pseudarthrosis in
Germany.
Literature:
1. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res.
1998(355 Suppl):S7-21. 
2. Schmidmaier G, Moghaddam A. [Long Bone Nonunion]. Z Orthop Unfall. 2015;153(6):659-
74; quiz 75-6. 
3. Hak DJ, Fitzpatrick D, Bishop JA, Marsh JL, Tilp S, Schnettler R, et al. Delayed union and
nonunions: epidemiology, clinical issues, and financial aspects. Injury. 2014;45 Suppl 2:S3-7. 
4. Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury.
2007;38 Suppl 4:S3-6. 
5. Romano CL, Logoluso N, Meani E, Romano D, De Vecchi E, Vassena C, et al. A comparative
study  of  the  use  of  bioactive  glass  S53P4  and  antibiotic-loaded  calcium-based  bone

substitutes in the treatment of chronic osteomyelitis: a retrospective comparative study.
Bone Joint J. 2014;96-B(6):845-50. 
6. Valimaki VV,  Aro HT. Molecular  basis for  action of bioactive  glasses as bone  graft
substitute. Scand J Surg. 2006;95(2):95-102. 
7. Lindfors N, Geurts J, Drago L, Arts JJ, Juutilainen V, Hyvonen P, et al. Antibacterial
Bioactive Glass, S53P4, for Chronic Bone Infections - A Multinational Study. Adv Exp Med
Biol. 2017. 
8. Lindfors NC, Hyvonen P, Nyyssonen M, Kirjavainen M, Kankare J, Gullichsen E, et al.
Bioactive  glass  S53P4  as  bone  graft  substitute  in  treatment  of  osteomyelitis.  Bone.
2010;47(2):212-8. 
9. van Gestel NA,  Geurts J, Hulsen DJ, van Rietbergen B, Hofmann S, Arts JJ. Clinical
Applications of S53P4 Bioactive Glass in Bone Healing and Osteomyelitic Treatment: A
Literature Review. BioMed research international. 2015;2015:684826. 
10. Kokubo T, Takadama  H. How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials. 2006;27(15):2907-15. 
11. Stoor P, Apajalahti S, Kontio R. Regeneration of Cystic Bone Cavities and Bone Defects
With Bioactive Glass S53P4 in the Upper and Lower Jaws. J Craniofac Surg. 2017;28(5):1197-
205. 
12. Stoor P, Pulkkinen J, Grenman R. Bioactive glass S53P4 in the filling of cavities in the
mastoid  cell  area  in  surgery  for  chronic  otitis  media.  Ann  Otol  Rhinol  Laryngol.
2010;119(6):377-82. 
13. Kankare J, Lindfors NC. Reconstruction of Vertebral Bone Defects using an Expandable
Replacement Device and Bioactive Glass S53P4 in the Treatment of Vertebral Osteomyelitis:
Three Patients and Three Pathogens. Scand J Surg. 2016. 
14. Fischer C, Frank M, Kunz P, Tanner M, Weber MA, Moghaddam A, et al. Dynamic
contrast-enhanced ultrasound (CEUS) after open and minimally invasive locked plating of
proximal humerus fractures. Injury. 2016;47(8):1725-31. 
15. Fischer C, Preubeta EM, Tanner M, Bruckner T, Krix M, Amarteifio E, et al. Dynamic
Contrast-Enhanced  Sonography  and  Dynamic  Contrast-Enhanced  Magnetic  Resonance
Imaging  for  Preoperative  Diagnosis  of  Infected  Nonunions.  J  Ultrasound  Med.
2016;35(5):933-42. 
16. Fischer C, Nissen M, Schmidmaier G, Bruckner T, Kauczor HU, Weber MA. Dynamic
contrast-enhanced magnetic resonance imaging (DCE-MRI) for the prediction of non-union
consolidation. Injury. 2017;48(2):357-63. 
17. Haubruck P, Kammerer A, Korff S, Apitz P, Xiao K, Buchler A, et al. The treatment of
nonunions with application of BMP-7 increases the expression pattern for angiogenic and
inflammable cytokines: a matched pair analysis. J Inflamm Res. 2016;9:155-65. 
18. Moghaddam A, Breier L, Haubruck P, Bender D, Biglari B, Wentzensen A, et al. Non-
unions treated with bone morphogenic protein 7: introducing the quantitative measurement
of human serum cytokine levels as  promising tool  in evaluation of adjunct non-union
therapy. J Inflamm (Lond). 2016;13:3.
3) Target criteria
To compare the clinical effectiveness of bioactive glass as a bone substitute in applied
Masquelet therapy with conventional Masquelet therapy in the treatment of large defect
pseudarthroses (2-5 cm3) of the tibia and femur.

Clinical and radiological investigation of the osteostimulative and osteoinductive potency of
bioactive glass in the treatment of large defect pseudarthrosis of tibia and femur.
To investigate the applicability of bioactive glass in the treatment of defect pseudarthrosis.
To investigate the angiogenic potential of bioactive glass in relation to the Masquelet
technique.
Primary  endpoints:  bony  consolidation  within  one  year  after  surgery  in  conventional
radiographic imaging in two planes (three of four cortices are bony bridged).
Secondary endpoints: Quality of life (SF36), perfusion (CEUS/DCE-MRI), pain (VAS), bony
consolidation within two years after surgery on conventional radiographic imaging in two
planes (three of four cortices are bony bridged), revisions, bony consolidation on CT one year
postoperatively, postoperative infections, cytokine expression patterns.
4) Test procedure
4.1 General description
The current study will compare two groups of patients. One group will receive standard
therapy for pseudarthrosis of the femur or tibia in terms of two-stage or
multi-stage  Masquelet  therapy.  In  the  second  group,  transplantation  of  autologous
cancellous bone is omitted in the second stage of Masquelet therapy and bioactive glass is
used instead. Clinical follow-up of pseudarthrosis patients is standardized in our department
and no additional examinations are performed as part of the study. As an extension of the
clinical follow-up examinations, only blood samples will be taken as part of the study to
determine  growth  factors,  infection  parameters,  and  blood  flow  markers.  The  total
collection volume of 32 ml of whole blood per follow-up is small.
4.2 Effects
Bioactive glass with the composition of SiO2, Na2O, CaO and P2O2 initiates growth of
tissues, stimulating bone growth (5-9). The effect of bioactive glass is based on the release of
ions (Si+, Na+, Ca2+) and their reaction with body fluids. After application in situ, these
surface reactions lead to the formation of a calcium phosphate layer that bonds with solid
materials such as bone (10). Numerous studies have demonstrated the clinical efficacy of
this mechanism in oral and maxillofacial surgery (11, 12), spine surgery (13), and trauma
surgery (5, 7-9, 13). Furthermore, numerous studies demonstrated the safety of the clinical
use of bioactive glass for filling bone defects (5, 7-9, 13). If these results are confirmed in the
current study, this represents a promising possibility for avoiding the additional intervention
required to obtain the bone graft in the future.
4.3 Adverse effects/risks
Bioactive glass (S53P4) is approved for the treatment of bone defects in pseudarthrosis in
Germany. Risks are assumed to be the usual surgical risks, but the indication for surgical
therapy will be based on purely medical reasons and will be completely independent of the
study. Furthermore, the patient may not respond to the therapy, which may result in

persistent pseudarthrosis. However, this risk exists for both standard therapy and Masquelet
therapy with bioactive glass as bone substitute. Follow-up examinations are performed
according to the same standard that is used for all patients treated in our clinic. Thus, there
is no undesirable burden on the patient. Only blood samples are taken in addition to the
standard. Possible undesirable effects of this blood collection correspond to the blood
collection that is normally performed in the clinic or by the general practitioner. The amount
of blood drawn includes 4 tubes, which is equivalent to 32 ml of blood. This has no relevant
influence on the total amount of blood. However, possible undesirable side effects may still
occur:
Pain at the puncture site, secondary bleeding, hematoma ("bruise"), infection (very rare),
vagal reaction with hypotension, nausea and possibly syncope ("fainting"), dizziness and
feeling of weakness after blood collection.
5) Study design
Investigator initiated Study
Clinical post-marketing observation study
Prospective randomized pilot clinical trial (RCT)
6) Randomization procedure
Balanced randomization, patients will be alternately assigned to treatment group G1 and G2
according to randomization time point.
7) Inclusion criteria
Patients suffering from pseudarthrosis of the tibia or femur with large bone defect (2-5cm3)
and operated by Masquelet technique.
8) Exclusion criteria
Patients under 18 years of age
Patients who do not consent to participate in the study.
Patients  who require  amputation  of  the  affected limb due  to persistent infections or
extensive soft tissue defects.
9) Discontinuation criteria
General: preliminary data or external data indicate unexpected risks to patients.
Individual: patients wish to discontinue their participation in the study
10) Study procedure

Patients suffering from pseudarthrosis with a large bone defect (2-5 cm3) will be treated
with the standard masquelet technique.
After  written  and  verbal  information  to  the  patients,  a  total  of  50  patients  will  be
randomized.  All  patients  will  receive  a  CEUS  ultrasound  examination  and  a  DCE  MRI
examination preoperatively to evaluate local perfusion.
Subsequently, in the first step of the Masquelet technique, inset implants are removed, the
pseudarthrosis is debrided, and a PMMA cement spacer is inserted after reduction. Either
internal or external fixation is performed depending on stability and soft tissue conditions.
During the first procedure, tissue samples are taken for microbiological examination. If
germs are positive, this procedure is repeated until all microbiological samples are germ-
free. Step two of the Masquelet technique is then performed six weeks postoperatively. The
PMMA cement is completely removed and the bone defect is filled with a combination of
autologous bone and a tricalcium phosphate matrix (G1) or bioactive glass (G2).
Subsequently, all patients receive the same standardized follow-up examinations. Initial
clinical  and  native-radiologic  follow-up  examinations  are  documented  two  days
postoperatively, and patients are discharged to outpatient care as soon as soft tissue
conditions permit. All patients also receive physical therapy in the outpatient setting. Clinical
and radiological follow-up examinations in our outpatient clinic are performed 6 weeks, 3
months, 6 months, 12 months, and 24 months postoperatively (see Table 1). Signs of
infection, range of motion of adjacent joints, pain (Visual Analog Scale), and weight-bearing
are obtained during the clinical examination. All radiological data are collected as part of the
clinical necessity; no additional radiological examinations are performed.
A questionnaire is used to document preoperative and 3,12 and 24 months postoperative
information on pain and range of motion, ability to work and quality of life.
General patient data such as occupation, BMI, profession, risk factors, prior medication,
prior surgery, and accident data will be documented preoperatively. One tube of special
EDTA and three serum tubes of blood (9 ml each) will be collected from all included patients
in chronological order on the above mentioned examination days. After this, the blood is
centrifuged within two hours and stored at -80°C. After this, the blood samples are analyzed
for  bone formation  and bone  resorption substances, infection values and markers for
perfusion. Local perfusion is assessed preoperatively and 12 weeks postoperatively by CEUS
and DCE-MRI.  Patients  in both  study groups  will be  divided according  to  clinical  and
radiological criteria into Therapy responders and therapy failures. After 12 months and after
24 months, all data will be statistically analyzed. A total of 50 patients will be included for a
period of two years, and the follow-up period will also be two years. The total study duration
is therefore four years.

preope
rative
2 days 
postop.
6 
weeks 
postop.
3 
months
postop.
6 
months
postop.
9
months
postop.
12
months
postop.
24
months
postop.
Clinical
examination
X
X
X
X
X
X
X
X
X-ray
X
X
X
X
X
X
X
 X
DCE-MRI
X
CT
X
CEUS
X
X
Questionnaire
SF-12
X
X
X
X
Blood sample
X
X
X
X
X
X
X
X
Table 1: All radiographs and CT scans were performed as part of the structured clinical
follow-up.
11) Statistical design
Primary and secondary endpoints will be evaluated categorically. The analysis will be non-
parametric using Wilcoxon test (p<= 0.05). All collected data will be analyzed and presented
descriptively.
12) Ethical and legal aspects
12.1 Ethical principles
The study will be conducted in accordance with the Declaration of Helsinki as amended in
October 2013.
12.1.1 Patient/subject information/consent form
Patients  will  be  informed  about  the  study  in  advance,  both  verbally  and  in  writing.
Participation is voluntary and patients will provide written informed consent.
12.2 Legal basis
The study will be conducted in accordance with the Declaration of Helsinki and the
Professional Code of Conduct for Physicians of the State Medical Association of Baden-
Württemberg in the respective current versions.
All X-ray examinations performed will be performed as part of routine clinical follow-up
examinations. No additional radiographs will be performed as part of the study.
12.2.1 Vote of the Ethics Committee

The study plan will be submitted to the Ethics Committee of the Heidelberg Medical Faculty
for review prior to study initiation. The inclusion of subjects/patients will not be started until
the written vote of the Ethics Committee has been received.
12.2.2 Data protection/view of original medical records
The  nam  es  of patients and  all  other confidential information are subject to medical
confidentiality and the provisions of the German Federal Data Protection Act (BDSG). Patient
data may only be passed on in anonymized form. Third parties will not be given access to
original medical records.
12.2.3 Data destruction in case of withdrawal
In the event of withdrawal from the study, all data not already published will be destroyed,
unless the patient has given prior written consent for the data already collected to be
evaluated.