Effectiveness of Two Bone Substitutes, Natural and Synthetic, in Preserving the Alveolar Ridge of Single-rooted Teeth: a Pilot Single-blind, Parallel Randomised Controlled Trial
Phase: NA
Conditions: Alveolar Ridge Augmentation
Sponsor: University of Central Lancashire

IRAS ID: 316275 V.2 04/05/2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
Effectiveness of two bone substitutes, natural and synthetic, in 
preserving the alveolar ridge of single-rooted teeth: a pilot single-
blind, parallel randomised controlled trial 
 
Date: 04/05/2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

IRAS ID: 316275 V.2 04/05/2023 
 
 
 
 
 
 
 
 
 
 
 
TITLE: 
Effectiveness of two bone substitutes, natural and 
synthetic, in preserving the alveolar ridge of single-rooted 
teeth: a pilot single-blind, parallel randomised controlled 
trial 
ACRONYM: 
BSNS (Bone Substitutes Natural Synthetic) 
 
Protocol version: 
1.0 
Date: 
15.11.2022 
 
 
 
Reviewed version   
2 
Date:  
 
 
04.05.2023 
 
 
IRAS project ID 
 
 
 
Trial registration 
TBC 
 
 
 
Study design 
Pilot single-blind, parallel randomised controlled trial 
 
 
 
Funding and support 
None 
 
 
 
Project CODE 
 
 
 
Confidentiality 
This document contains confidential information that must 
not be disclosed to anyone other than the Sponsor, the 
Investigators Team, host organisation (s), and the Research 
Ethics Committee members unless authorised to do so. 

IRAS ID: 316275 V.2 04/05/2023 
 
 
 
 
 
 
Contents 
Lay Summary ..........................................................................................................................i 
Abstract .................................................................................................................................. ii 
Introduction ......................................................................................................................... ii 
Methods .............................................................................................................................. ii 
Discussion .......................................................................................................................... ii 
Ethics and dissemination ................................................................................................... iii 
Background ........................................................................................................................... 1 
What is the problem to be addressed? ............................................................................... 1 
Aim ........................................................................................................................................ 2 
Key research questions ......................................................................................................... 2 
Trial design ........................................................................................................................... 3 
Methods ................................................................................................................................ 3 
Study setting ...................................................................................................................... 3 
Eligibility criteria for participants ......................................................................................... 3 
Intervention ........................................................................................................................ 4 
Management of the bone substitutes.................................................................................. 4 
Clinical procedure .................................................................................................................. 5 
Management of tissue samples in the dental clinic ............................................................. 5 
Management of tissue samples for Histology ..................................................................... 6 
Packaging of tissue samples for Histology ......................................................................... 6 
Labelling of tissue samples for Histology ............................................................................ 6 
Transport documentation of tissue samples for Histology ................................................... 6 
Tracking tissue samples for Histology ................................................................................ 6 
Laboratory processing of tissue samples for Histology ....................................................... 7 
Management of tissue samples for PCR ............................................................................ 7 
Packaging of tissue samples for PCR ................................................................................ 7 
Labelling of tissue samples for PCR ................................................................................... 7 
Transport documentation and tracking tissue samples for PCR .......................................... 8 
Laboratory processing of tissue samples for PCR .............................................................. 8 

IRAS ID: 316275 V.2 04/05/2023 
 
Criteria for discontinuing or modifying allocated interventions ........................................... 10 
Strategies for monitoring and improving protocol adherence ............................................ 11 
Outcomes ........................................................................................................................ 11 
Sample size ..................................................................................................................... 11 
Clinician-patient ratio ........................................................................................................ 12 
Randomisation, sequence generation, allocation and blinding .................................. 12 
Data collection ................................................................................................................. 13 
Data management ........................................................................................................... 13 
Statistical methods ........................................................................................................ 14 
Primary outcome .............................................................................................................. 14 
Secondary outcomes ....................................................................................................... 14 
Data monitoring ............................................................................................................... 15 
Risk and safety issues ..................................................................................................... 15 
Harm ................................................................................................................................ 15 
Auditing ........................................................................................................................... 15 
Research ethics approval ................................................................................................. 15 
Protocol amendments ...................................................................................................... 15 
Consent, invitation and confidentiality .................................................................................. 16 
Declaration of interest ...................................................................................................... 16 
Dissemination policy ........................................................................................................ 16 
Ancillary post-trial care ........................................................................................................ 16 
Patient and Public Involvement (PPI) ................................................................................... 17 
Discussion ........................................................................................................................... 17 
Conclusion .......................................................................................................................... 18 
Protocol version .................................................................................................................. 18 
Funding ............................................................................................................................... 18 
Acknowledgements ............................................................................................................. 18 
Author contributions ............................................................................................................. 18 
References .......................................................................................................................... 19 
Appendix ............................................................................................................................. 23 
Appendix 1 ASA physical status classification system ...................................................... 23 
Appendix 2 Instructions for the use of Bonalive ................................................................ 25 

IRAS ID: 316275 V.2 04/05/2023 
 
Appendix 3 Instructions for the use of Bio-Oss® ........................................................................................................ 25 
Appendix 4 Packaging instruction .................................................................................... 26 
Appendix 5 Biological substance category label ............................................................... 28 
Appendix 7 Dry ice label .................................................................................................. 29 
Appendix 8 Email from MHRA .......................................................................................... 31 
 
 
 
 
KEY STUDY CONTACTS 
 
Chief Investigator 
Ihtesham ur Rehman 
(iurehman@uclan.ac.uk) 
Principal investigator 
Fadi N Barrak (FNBarrak@uclan.ac.uk) 
Sponsor 
University of Central Lancashire (UCLan) 
Joint-sponsor(s)/co-sponsor(s) 
n/a 
Funder(s) 
n/a 
Key Protocol contributors 
Fadi N Barrak, Aderonke Ajiboye, R Jones, 
Seiwi Li, Andrea Manfrin 
 
 
Study summary 
 
Study title 
Effectiveness of bone substitutes, natural 
and synthetic in preserving the alveolar ridge 
of single-rooted teeth: a p i l o t  single-
blind, parallel randomised 
controlled trial 
Internal ref.no. (or short title) 
BSNS 
(Bone 
Substitutes 
Natural 
vs 
Synthetic) 
Study design 
Pilot single-blind, parallel randomised 
controlled trial 
Study participants 
Adult patients who require extraction of 
single-rooted teeth 
Planned size of Sample 
34 patients, 2-4 dental practices 
Planned study Period 
12 months with 24-week follow-up 
Research Questions/Aim(s) 
Research 
questions: 
What 
are 
the 
differences between natural and synthetic 

IRAS ID: 316275 V.2 04/05/2023 
 
 
bone substitutes in preserving the alveolar 
ridge dimensions based on: 
i. 
 Changes in the width of the alveolar 
ridge (expressed in millimetres) from 
baseline to 24-week follow-up? 
ii. 
Changes in height of the alveolar 
ridge (expressed in millimetres) from 
baseline to 24-week follow-up? 
The two changes will be assessed 
with either an intraoral scanner or 
impressions 
and 
clinical 
photographs. 
iii. 
 Changes in the vertical crestal bone 
level assessed in millimetres at 24- 
week follow-up? 
iv. 
Presence 
of 
bone 
around 
the 
alveolar ridge to be assessed using 
biopsy (up to 3mm diameter by 8mm 
length) 
for 
histology 
(qualitative 
assessment of bone infiltration) and 
PCR to determine the concentration 
(in nanograms) of gene expression 
for bone markers at 24-week follow- 
up? 
Aim: To investigate the effectiveness of two 
low substitution rate bone substitutes, 
natural and synthetic in preserving the 
alveolar ridge of single- rooted teeth 
following extraction. 
 
 
FUNDING AND SUPPORT IN KIND 
 
FUNDER(S) 
n/a 

IRAS ID: 316275 V.2 04/05/2023 
 
 
 
ROLE OF STUDY SPONSOR AND FUNDER 
The sponsor, UCLan, takes on overall responsibility for proportionate, effective arrangements being in 
place to set up, run and report a research project 
 
 
Study coordination 
 
Dr Fadi N Barrak, the Principal investigator (PI), will be responsible for researching the site 
under the Chief investigator's supervision 

IRAS ID: 316275 V2 04/05/202 
i 
 
 
 
Lay Summary 
 
After a tooth is extracted, the bone that supports and surrounds the tooth can considerably 
shrink. This loss of bone volume around the missing tooth can make future treatment with 
dentures or dental implants more challenging. It may even necessitate surgical procedures to 
increase the bone volume in the area where the extraction was carried out and replace the 
lost bone. Therefore, it is vital to consider the preservation of the bone that supports the tooth 
to limit the reduction of bone volume after an extraction. Bone substitutes can be used to pack 
the extraction socket to   preserve the space. 
 
The most commonly used bone substitutes in dentistry are cow bone. However, as people are 
becoming less inclined to use animal derivates, synthetic bone substitutes could become a 
feasible alternative. This study aims to evaluate the efficacy of one of these synthetic bone 
substitutes – bioactive glass (BAG) – in preserving the bone that supports a tooth after an 
extraction. 
 
A few synthetic alternatives to cow bone are already available on the market. However, 
their properties are not as good as traditional cow bone. They are resorbed much more quickly, 
and therefore, in the long term, they fail to maintain the shape and volume of the bone in the 
extraction socket. What is different about BAG is the fact that it not only has similar long-term 
preservation features as cow bone, but it also has the potential to surpass cow bone because 
of its unique antibacterial activity. 
 
The study will be conducted at the University of Central Lancashire, Imperial College London 
and dental clinics in Midlands, Southeast and Northwest England. We will recruit 34 patients 
who require tooth extractions and divide them into two equal groups. Patients in  group A will 
receive BAG substitute, while those in the group B will receive the natural bone substitute. For 
both groups, the bone substitute will be inserted immediately after an extraction, and the 
socket will be sealed with a membrane sutured in place. Patients in both groups will be 
reviewed after 2 weeks to remove the suture. Patients will have another review at 24 weeks, 
during which the researcher will take a scan or mould, a photograph, and a small biopsy 
sample of the area where the bone substitute was placed. Measurements obtained at the 24-
week follow-up will be used to evaluate the effectiveness and differences between natural and 
synthetic bone substitutes in preserving the bone supporting a tooth after an extraction. 

IRAS ID: 316275 V2 04/05/202 
ii 
 
 
 
The benefit of this study lies in the possibility of having a synthetic and antibacterial alternative 
to a naturally derived bone substitute. The results will inform the design of a full randomised 
controlled trial. 
 
Abstract 
Introduction 
Using bone substitutes to preserve the alveolar ridge of a missing tooth is important for 
ensuring the retention and stability of the prosthesis used as a replacement. In dentistry, the 
most used bone substitutes are bovine derived. However, the movement toward a more 
environmentally accountable society has created a need for synthetic bone substitutes which 
are not derived from animals. Therefore, this study aims to evaluate the effectiveness of a 
bovine-derived bone substitute and bioactive glass (BAG) derived bone substitute in 
preserving the alveolar bone of a tooth after an extraction. 
Methods 
A pilot single-blind two-arm parallel randomised control trial will be conducted in dental clinics 
in the Midlands, Southeast and Northwest England. A total of 34 adult patients, who require 
the extraction of a single-rooted tooth, will be randomly assigned to experimental and control 
group at a  1:1 ratio.  
After tooth extraction, the bone substitute will be introduced into the socket; the control group 
A will receive the bovine-derived bone the intervention group B the bioactive glass derived 
bone. All patients will be reviewed after 2 weeks for suture removal. Patients’ follow-up will be 
24 weeks after the procedure. Outcome data will be collected at baseline and at 24 weeks. The 
primary outcome is the effectiveness of bioactive glass derived bone  in preserving the alveolar 
ridge dimensions compared to bovine-derived bone. This will be assessed by measuring 
changes in the width of the alveolar ridge from baseline (augmentation) to 24-week post 
augmentation using a linear regression model including baseline measurements as 
covariates. Secondary outcomes are: 
• 
Changes in the height of the alveolar ridge from augmentation to 24-week post 
augmentation 
• 
Changes in vertical crestal bone level at 24-week post augmentation 
• 
Presence of bone markers at 24-week post augmentation 
• 
Qualitative Bone infiltration at 24-week post augmentation 
 
 
Discussion 
 

IRAS ID: 316275 V2 04/05/202 
iii 
 
 
The results of this pilot study will inform the design and implementation of a full scale 
randomised controlled trial to investigate the effectiveness of bioactive glass derived bone 
compared with bovine derived bone in preserving the alveolar bone of a tooth after an 
extraction. 
 

IRAS ID: 316275 V2 04/05/202 
iv 
 
 
 
Ethics and dissemination 
Ethics approval was obtained from the University of Central Lancashire xxxxxxx. Results will 
be published in peer-review and professional journals, presented at scientific conferences, 
and disseminated to service users and their families via media. 
 
Trial registration number: XXXXX 

IRAS ID: 316275 V.2 04/1/2022 
1 
 
 
 
 
Effectiveness of two bone substitutes, natural and synthetic, in preserving the alveolar 
ridge of single-rooted teeth: a single-blind, parallel randomised controlled trial 
 
Background 
What is the problem to be addressed? 
Implant dentistry has expanded dramatically over the last decade globally and is forecast to 
continue to do so for the foreseeable future, not least due to the increasing proportion of over 
60-year-olds within the population. For example, a study conducted in 2018 reported that the 
prevalence of dental implants in the United States increased from 0.7% in 1999 to 5.7% in 
2015(1) . Elani et al. (1)  also noted that the highest absolute growth in prevalence (12.9%) 
was among the age group 65 to 74 years. Implant treatments are varied and approximately 
50% involve the use of bone substitutes to compensate for the jawbone deficiencies and 
enable implant fixtures' placement (2,3). The use of bone substitutes has grown in parallel with 
implants. These substitutes are used routinely for ‘guided bone regeneration’, sinus 
augmentation, treating peri-implantitis (infection around implants where bone loss occurs), and 
ridge preservation (4–6). The latter involves placing bone substitute in a fresh extraction 
socket, immediately after the tooth is removed in order to preserve the jawbone ridge for future 
implant placement (7,8) . 
Bone substitute materials available in the market include allografts (human donated bone), 
xenografts (from a different species, which can be plant or animal) and alloplasts (synthetic) 
(6). Historically, the most commonly used material has been xenograft of bovine origin due to 
its ease of handling and extensive research to support its safety and efficiency in maintaining 
bone volume. In a 5-year prospective study, Ozkan et al (9).  reported that using bovine-derived 
bone in 1-stage sinus augmentation resulted in sufficient quality and volume of bone for 
implant placement and a 100% implant survival rate after the follow-up period. 
Despite the effectiveness of bovine xenografts, the movement towards a more environmentally 
accountable society has led to patients finding animal-derived bone substitutes less 
acceptable (10,11). As clinicians, we also must consider this important aspect of our work and 
be accountable in terms of our material choices while bearing in mind the environment and 
patient safety and benefit. 
The current synthetic products on the market work well in terms of safety; however, they are 
mainly calcium phosphate and sulphate based and have a high substitution rate. That means 
that they are resorbed and remodelled quickly when compared to the bovine bone substitutes 

IRAS ID: 316275 V.2 04/1/2022 
2 
 
 
 
(12) The allografts (human origin) also have a high substitution rate. It is important to have a 
low substitution rate to maintain the shape and volume of the jaw ridge for the long term to 
have a stable result. 
Bioactive glass synthetic bone substitute (Bonalive®) is a material that has been shown to have 
a low substitution rate and antibacterial properties. It has been used successfully in the 
orthopaedics, craniofacial and ear nose and throat (ENT) specialities for the treatment of 
osteomyelitis, sinusitis and bony cyst cavities (13,14). There have also been a few 
maxillofacial surgery case reports (15,16) on its usage in the jaws. However, to the best of our 
knowledge, the applicability of this material in implant dentistry has not been reported in the 
literature. 
 
Aim 
To investigate the effectiveness of two low substitution rate bone substitutes, natural (Bio- 
Oss®) and synthetic (Bonalive®) in preserving the alveolar ridge of single-rooted teeth 
following extraction. 
 
Key research questions 
Is there a difference between natural (Bio-Oss®) and synthetic (Bonalive®) bone substitutes in 
preserving the alveolar ridge dimensions based on: 
I. 
Changes in the width of the alveolar ridge (expressed in millimetres) from 
augmentation (baseline) to 24-week follow-up (post augmentation). 
II. 
Changes in the height of the alveolar ridge (expressed in millimetres) from 
augmentation (baseline) to 24-week follow-up (post augmentation). 
Both changes will be assessed using intra-oral scanners (this does not involve 
radiographs) or impressions with clinical photographs. 
III. 
Changes in the vertical crestal bone level assessed in millimetres, using the periapical 
radiographs taken pre-extraction and at 24-week follow-up and intra-oral scanners (this 
does not involve radiographs) or impressions with clinical photographs 
IV. 
Presence of bone around the alveolar ridge assessed using biopsy (up to 3mm 
diameter by 8mm length) of the centre of the augmented socket. The biopsy sample 
will be used for histology (qualitative assessment of bone infiltration) and PCR to 
determine the concentration (in nanograms) of gene expression for bone markers 
(including osteopontin, osteocalcin) at 24-week follow-up (post augmentation). 

IRAS ID: 316275 V.2 04/1/2022 
3 
 
 
 
Trial design 
It is a pilot single-blind two-arm parallel randomised control trial that aims to assess the 
differences between two bone substitutes, natural versus synthetic. The study will mimic the 
routine practice, and patients will be randomly allocated to receive either the natural (control) 
or synthetic (intervention) bone substitutes. The patient follow-up period will be 24 weeks from 
the baseline data collection point. 
 
Methods 
Study setting 
The study will be conducted in dental clinics in the Midlands, Southeast England and 
Northwest England. 
 
 
Eligibility criteria for participants 
Inclusion criteria 
Adult patients (age >18) in need of extraction of a single-rooted tooth attending the clinics 
selected for the study 
Single units in a dentate patient 
 
Non-surgical extraction – no flap raised, and no bone removed 
Intact socket walls post-extraction 
Bleeding sockets 
 
Stable periodontal health 
 
ASA Class I or II patients. The American Society of Anaesthesiologists (ASA) classification is 
a system of physical status evaluation developed to offer clinicians a simple categorisation of 
a patient's physiological status to help predict operative risks (Appendix 1). 
 
 
Exclusion Criteria 
 
Multiple adjacent extractions 
 
Presence of active periodontal disease 
 
Socket walls not intact - >50% bone loss in any of the four walls 

IRAS ID: 316275 V.2 04/1/2022 
4 
 
 
 
Sclerotic sockets post-extraction – the socket does not fill up with blood post-extraction 
Denture wearer – the extraction socket site under the load of a denture 
Patients on medication that can affect bone healing e.g., bisphosphonates (oral or 
intravenous), selective serotonin reuptake inhibitors (SSRI), methotrexate, proton pump 
inhibitors (PPI) 
Uncontrolled diabetes 
Smokers 
Immunosuppressed Patients 
Patients with a history of myocardial Infarction in the last year 
ASA Class > II patients (17)(Appendix 1) 
 
Intervention 
The test bone substitute is Bonalive®, bioactive glass S53P4, which contains SiO2, Na2O, CaO, 
and P2O5 (granule size 0.5-0.8mm) (18) Bonalive® is osteoconductive, meaning that it has the 
ability of promoting bone growth across the granules and the grafting area and slowly replace 
it with new bone over time. Bonalive® is osteostimulative and has antibacterial properties (19). 
 
Comparator 
The comparator is Bio-Oss®, deproteinized bovine bone granules (granule size 0.25- 1mm) 
(20). Bio-Oss® is osteoconductive, which means it acts as a scaffold only for new bone to grow 
(21,22). 
 
Both products have a low substitution rate and are expected to have similar ridge support and 
shape maintenance capability. 
 
Management of the bone substitutes 
The bone substitutes will be used according to the manufacturers’ instructions and protocols 
(Appendix 2 and 3). The materials are supplied in sealed sterile packaging with clearly 
displayed expiry dates, LOT numbers and CE marks. The materials’ LOT numbers and expiry 
dates will be recorded on the patient records and on a separate ‘Surgery Logbook’ with an 
identification number for the patient traceable to the appropriate batch of materials. 

IRAS ID: 316275 V.2 04/1/2022 
5 
 
 
 
The use of the material will follow standard surgical protocols and excess material will be 
discarded according to clinical waste guidelines. 
 
Clinical procedure 
The bone substitute will be used according to the ridge preservation protocol, which will be 
standardised between the different centres. As part of standard clinical practice, a periapical 
radiograph will be taken before an extraction. Following the removal of a tooth, the inflamed 
area of the socket will be curetted to ensure a ‘clean’ socket. The case will be excluded if: 
• 
The socket walls are very sclerotic with a lack of fresh bleeding, which is essential for 
healing. 
• 
The socket walls are damaged. The limit is a loss of 50% or more of one of the walls. 
The assessment of the extraction socket will be done by the clinicians performing the 
procedures (23). 
The bone substitute will then be introduced using a standard sterile protocol, with gentle 
packing of the material in the sockets to ensure the material reaches the apex of the socket 
but is not condensed with pressure. The socket will then be sealed with a collagen matrix with 
non-resorbable sutures. Review appointment for suture removal will be required 2 weeks post- 
procedure. 
Patients will be advised not to fly for two weeks post-procedure or until after the clinicians have 
reviewed them. Intra-oral scans or impressions with clinical photographs will also be taken on 
the day the procedure is carried out. The follow-up will take place 24 weeks post-procedure, 
when a PA radiograph, intra-oral scans or impressions, clinical photographs and a minimally 
invasive trephine biopsy will be carried out. 
Management of tissue samples in the dental clinic 
 
Each sample taken will be split into two segments (one for histology and the other for PCR). 
The segment for histology will be fixed in formaldehyde, and the one for PCR analyses will be 
snap-frozen in liquid nitrogen (or dry ice) and placed in -80C (or -20C, in RNAlater®-ICE) 
freezer for storage. The samples will be transported from the private dental clinics to the 
Imperial College Healthcare Tissue Bank (ICHTB) as they are collected. 

IRAS ID: 316275 V.2 04/1/2022 
6 
 
 
 
Management of tissue samples for Histology 
 
Packaging of tissue samples for Histology 
 
The packaging will adhere to ADR Packing Instruction 650 for BIOLOGICAL SUBSTANCE, 
CATEGORY B (Appendix 4). This will consist of three components: 
 
• 
A primary package which will contain the sample. 
• 
A secondary package which will contain the primary package and sufficient absorbent 
material to soak up any spillage that occurs. 
• 
A tertiary/outer packaging which will be rigid and have at least one surface with a 
minimum dimension of 100mm x 100mm 
 
Labelling of tissue samples for Histology 
 
• 
Each package will be clearly labelled with the delivery address and sender’s details. It 
will also have emergency contact details, including a named person and telephone 
number for the sender (the private dental clinic) and recipient (ICHTB). 
• 
The outer package will be clearly marked with BIOLOGICAL SUBSTANCE and 
CATEGORY B letters at least 6mm high. This will be displayed adjacent to the UN3373 
label. 
• 
The UN3373 label will be affixed to the outside packaging as a square set at an angle 
of 45° (e.g., diamond shaped), with each side having a length of at least 50 mm, the 
width of the line will be at least 2mm and the letters and numbers at least 6mm high 
(see UN3373 label example in Appendix 5). The background of the mark will have a 
contrasting colour to the surface of the package. 
 
Transport documentation of tissue samples for Histology 
 
Paperwork with a contents list will be included within the package between the secondary and 
the outer packaging. The paperwork will be placed in waterproof packaging and, if handwritten, 
it will be in permanent ink. 
 
Tracking tissue samples for Histology 
 
The management of the tissue samples at ICHTB will be both ethical and compliant with the 
Human Tissue Authority (HTA) regulations. (See ICHTB’s HTA license number in appendix 

IRAS ID: 316275 V.2 04/1/2022 
7 
 
 
 
6). For all samples, a record containing pseudo-anonymised information about the donor, the 
samples taken, the type of consent obtained, and the surgical procedure used to collect the 
samples will be created. This information will be recorded in an online database which is 
compliant with the General Data Protection Registration legislation. 
 
 
Laboratory processing of tissue samples for Histology 
 
The procedure for processing the sample in the laboratory will be similar to that used in a study 
by Shi et al. (24). Samples will be dehydrated through a series of increasing concentrations of 
ethanol and embedded in white acrylic resin for sectioning. The sample blocks will be ground 
successively with (800, 2000 and 4000 grit) grinding paper to expose and polish the bone/hard 
tissue. The polished surface will then be glued onto an acrylic disc. The tissue block will then 
be ground with the same series of grinding paper resulting in a sample of approximately 50-
100 µm in thickness. According to the manufacturer's instructions, sections will be stained with 
Gill’s Haematoxylin and Eosin. Stained samples will then be examined and imaged under a 
microscope. 
Management of tissue samples for PCR. 
Packaging of tissue samples for PCR 
 
Samples will be placed in a primary package (RNAlater®-ICE in leak-proof Cellstor® biopsy 
pots) and put in a secondary package (a BiTran® double-zip lock bag), transported in a dry 
ice-filled styrofoam box. The outer packaging will allow for carbon dioxide gas release to avoid 
gas accumulation and potential rupturing of packaging or explosion. 
 
Labelling of tissue samples for PCR 
 
 
• 
Each package will be clearly labelled with the delivery address and sender’s details. It 
will also have emergency contact details, including a named person and telephone 
number for where the package is being sent from and where it is going. 
• 
A Class 9 - Miscellaneous Dangerous Goods label with the words DRY ICE next to it, 
will be clearly visible on the outside packaging. (See UN1845 label example in 
Appendix 7). 

IRAS ID: 316275 V.2 04/1/2022 
8 
 
 
 
Transport documentation and tracking tissue samples for PCR 
 
The protocols for transport documentation and tracking of the tissue samples will be the same 
as that for samples taken for histology. 
 
Laboratory processing of tissue samples for PCR 
 
The procedure for processing the sample in the laboratory will be similar to that used in a study 
by Li et al. (25) Frozen tissue samples will be thawed in RNAlater®-ICE and lysed for RNA 
extraction using Qiagen RNeasy kit following the manufacturer’s instructions. Following 
treatment with DNase-1 reagent and reverse-transcription of RNA samples using the 
SuperScript® VILOTM cDNA synthesis. SYBR green-based qPCR assays will be performed 
to analyse osteogenic gene expression, including Runx2, alkaline phosphatase, osteopontin 
and osteocalcin. The relative transcript levels of genes of interest will be analysed using the 
comparative CT (ÄÄCT) method. The range of relative transcript levels of the genes of interest 
will be presented as bar graphs, and statistical analysis will be performed at the level of ÄÄCT. 

IRAS ID: 316275 V.2 04/1/2022 
9 
 
 
 
 
 
 
Table 1 Schedule of enrolment, intervention and assessments 
 
Study period 
Enrolment 
Allocation 
Post-allocation 
Close- 
out 
Timepoint (weeks) 
-W0 
W0 
W0 
W2 
W24 
Enrolment 
Eligibility 
screen 
for 
patients 
x 
Informed consent from 
patients 
x 
List of other procedures 
x 
Randomisation 
of 
patients 
x 
Interventions 
Synthetic bone 
x 
Natural bone 
x 
Suture removal 
x 
Assessments 
(Outcomes) 
Width 
x 
x 
Height 
x 
x 
Crestal bone 
x 
Histology 
x 
Bone Markers 
x 

IRAS ID: 316275 V.2 04/1/2022 
10 
 
 
 
Figure 1 CONSORT study flowchart 
 
 
 
 
Criteria for discontinuing or modifying allocated interventions 
 
• 
Participant withdraws consent 
• 
The trial is discontinued 
• 
Participant has an adverse reaction 
 
The reasons for discontinuation will be documented (see Standard Operating Procedures). 

IRAS ID: 316275 V.2 04/1/2022 
11 
 
 
 
Strategies for monitoring and improving protocol adherence 
There are some measures that clinicians could adopt to improve patients' adherence to the 
research protocol, such as telephone calls, text reminders, and social support to educate 
patients. In addition, clinicians will have to create a welcoming, non-judgmental and accepting 
environment; educate patients about their role as research participants; establish a routine 
while maintaining flexibility; provide incentives for participation, parking spaces, and 
videoconferences. 
Outcomes 
Primary outcome 
 
The primary outcome is the difference between the two low substitution rate bone substitutes, 
natural (Bio-Oss®) and synthetic (Bonalive®), in preserving the alveolar ridge dimensions 
assessed by changes in the width (expressed in millimetres) from augmentation (baseline) to 
24-week post augmentation. Assessment will be done using intra-oral scanners (this does not 
involve radiographs) or impressions and clinical photographs. 
Secondary outcomes 
 
• 
Changes in height (expressed in millimetres) from augmentation (baseline) to 24-week 
post augmentation. Assessment will be done using the periapical radiographs taken 
pre-extraction and at 24-week follow-up and intra-oral scanners (this does not involve 
radiographs) or impressions and clinical photographs. 
• 
Changes in the vertical crestal bone level (expressed in millimetres) assessed with 
periapical radiographs taken pre-extraction and 24-week post augmentation. The 
technique used for this assessment will be the same as that used by Solakoglu et 
al.(26) to evaluate changes in the alveolar bone crest in similar research. 
• 
Presence of bone around the alveolar ridge assessed using a biopsy, histology 
(qualitative assessment of bone infiltration) and PCR to determine the concentration 
(in nanograms) of gene expression for bone markers (including osteopontin, 
osteocalcin) at 24-week post augmentation. 
 
Sample size 
According to Charles et al. (27) four factors are required to calculate the sample size: 
significance level, power, difference between groups and standard deviation. However, 
considering the limited availability of information in this area (only one study was identified 
conducted by Rignon-Bret et al.(28)  

IRAS ID: 316275 V.2 04/1/2022 
12 
 
 
 
 The research team, supported by the advice provided by statisticians, deiced to conduct a 
pilot study. Even though a pilot study does not require sample size calculation using the 
aforementioned four factors,(29) 15 to 20 participants per group are required to ensure the 
scientific validity of the pilot study results (30) . More recently, further information has been 
provided for the validity of the results of pilot RCT indicating a wider range of participants (31). 
Therefore, we used a pragmatic approach informed by the literature and we will recruit a total 
of 34 participants with 17 per group. The results of the pilot study will generate the four factors 
to calculate the sample size for the full-scale RCT.  
  

IRAS ID: 316275 V.2 04/1/2022 
13 
 
 
 
Recruitment 
 
Recruitment will be focused on private dental clinics in the Midlands, Southeast England and 
Northwest England 
Clinicians: Experienced Implant surgeons at centres approved by lead researchers. Approval 
will be based on appropriate clinical setup, clinician experience and patient volume. 
Patients: Patients who meet the inclusion criteria will be invited to take part in the study. They 
will consent to using either of the bone substitute materials and be randomly allocated to a 
group. 
Recruitment will start as soon as ethics approval has been granted. 
 
The recruitment strategy will increase potential participants’ awareness of the health problem 
being studied and its potential impact on their health. 
Clinician-patient ratio 
The patients will be spread across 2-4 surgeries to ensure we meet the target sample size in 
a few months. 
Randomisation, sequence generation, allocation and blinding 
 
The randomisation, sequence generation and allocation concealment will be performed using 
the sealed envelope online system (https://www.sealedenvelope.com/) (32) . The 
randomisation will be 1:1. The patient will be  the unit of randomisation and intervention. 
 

IRAS ID: 316275 V.2 04/1/2022 
14 
 
 
 
 
 
All patients receiving the bone substitutes will be blinded to the treatment in our study. 
However, patients will know which bone substitute they have received after their final review 
at 24-week follow-up. 
Data collection 
Clinicians will collect data at baseline and at 24-week follow-up using a paper template. Then 
data will be transferred into Excel and SPSS and prepared for the data analysis. All paper 
records, except consent forms, will be destroyed at patients’ last visit. Secure and irreversible 
destruction processes will include shredding and disposal of records using the University’s 
confidential waste service. 
Data management 
The procedure will be conducted using the data management template provided by UKRI. 
Data will be managed following the procedure used in previous studies (26,28). Input data will  
 
be saved and stored on a password-protected system. Only individuals authorised by the CI 
will be allowed to access the data. Paper data, such as Patients’ informed consent will be kept 
in a locked cabinet by the clinicians. 

IRAS ID: 316275 V.2 04/1/2022 
15 
 
 
 
Statistical methods 
The primary analysis will be the intention-to-treat (ITT), including all randomised participants 
in the assigned group, regardless of their adherence to the protocol or their withdrawal. 
Missing data will be assessed and treated using multiple imputations if missing at random 
(MAR). 
To check for normality, each variable will be analysed using the Shapiro-Wilk test. 
 
Primary outcome 
The primary outcome is the difference between the two bone substitutes, natural (Bio-Oss®) 
and synthetic (Bonalive®), in preserving the alveolar ridge dimensions assessed by changes 
in the width of the alveolar ridge (expressed in millimetres) from augmentation (baseline) to 
24-week post augmentation. It is expected to be a continuous variable, therefore, the 
comparison between groups at 24 weeks will be performed using a linear regression model 
(ANCOVA), including the baseline measurements as the covariates. The recent literature 
suggests using of ANCOVA as the statistical method of choice for the analysis of intervention 
effect and adjustment for baseline variables for three reasons: efficiency, precision and power 
(33).  
Secondary outcomes 
• 
The changes in the height of the alveolar ridge (expressed in millimetres) from grafting 
(baseline) to 24-week follow-up (post grafting) assessed using either intra-oral scanners 
or impressions and clinical photographs. It is expected to be a continuous variable; 
therefore, the comparison between groups at week 24 will be performed using the 
unpaired t-test for a normally distributed variable or the Mann-Whitney U test for non- 
normally distributed. 
• 
The changes in vertical crestal bone levels between the groups (expressed in millimetres) 
will be compared using an unpaired t-test or Mann-Whitney U test according to the 
variable distribution. This will be assessed using radiographs pre-extraction and at 24- 
week follow-up. 
• 
Presence of bone will be assessed using biopsy used for histology (qualitative 
assessment of bone infiltration) and PCR to determine the concentration (in nanograms) 
of gene expression of the bone markers (including osteopontin and osteocalcin). 

IRAS ID: 316275 V.2 04/1/2022 
16 
 
 
 
• 
The comparison of the concentration of gene expression between the groups at 24-week 
will be performed using the unpaired t-test for a normally distributed variable or the Mann- 
Whitney U test for non-normally distributed 
 
Data monitoring 
This trial is designed to minimise the risk, as demonstrated in the previous trials by Rignon- 
Bret et al. (28)and Solakoglu et al. (26). Therefore, no formal committee has been organised, 
and no interim analysis of the impact of the intervention has been planned. 
Risk and safety issues 
The risk of allergy, infection and lack of bone integration will be monitored by the clinicians 
during the study keeping in contact with their patients. 
Harm 
We do not envisage any specific harm from these products, which are approved and in clinical 
use already, other than risks of potential complications from surgical procedure. This will be 
minimised through careful and appropriate case selection (healthy ASA Class I and II), surgical 
procedures with standard precautions for infection control and pre- and post-operative care. 
Additionally, the principal investigator enquired the Medical and Healthcare Products 
Regulatory Agency (MHRA) regarding the use of these bone substitutes in the study. The 
MHRA replied that “as you are using the medical devices on human subjects within its CE 
marked intended use, as per the manufacturers IFU and the intention of the study is not to 
generate data to change or extend the indications of the CE marked device, you do not need 
to notify MHRA of this study” (Appendix 8). 
Auditing 
No audit has been planned at this time. 
Research ethics approval 
Ethics approval will be sought from the Health Ethics Review Panel at the University of Central 
Lancashire (UCLan) 
Protocol amendments 
We are not expecting to make any changes to the eligibility criteria, outcomes or analyses 
during our study 

IRAS ID: 316275 V.2 04/1/2022 
17 
 
 
 
Consent, invitation and confidentiality 
All documentation related to information and consent for clinicians and patients has been 
enclosed in the protocol and approved by the UCLan ethics committees. The procedures 
followed for consent and confidentiality are described in the following paragraphs. 
 
Informed consent 
Clinicians participating in the study will recruit their patients and obtain consent from them. 
 
 
Patients’ recruitment and informed consent 
After assessing patients’ eligibility for the study, clinicians will provide an information letter 
and consent form to each patient, who will get a week to consider their participation. The 
clinicians will submit all signed consent forms to the PI so that they can be stored in the PI’s 
office in a locked cabinet located within the School of Dentistry. 
The baseline information will be transferred onto an Excel spreadsheet and stored 
electronically in a password protected UCLan computer. Anyone who is deemed unsuitable to 
participate will be offered a copy of their record and the electronic record will be destroyed in 
line with the University’s confidential procedure. 
 
Declaration of interest 
None 
 
Dissemination policy 
The dissemination of the study will begin immediately with the publication of the protocol. The 
results of this trial will be presented at national and international conferences. They will be 
submitted as scientific manuscripts to peer-reviewed journals. The trial results aim to inform 
patients, policymakers, and all other stakeholders that might benefit from the results. 
The results of the trial will be disseminated to service users and their families via media, to 
healthcare professionals via professional training and meetings, and researchers via 
conferences and publications. The publications generated by this study may be used as 
training materials for dentists with an interest in implantology. 
 
Ancillary post-trial care 
We are not envisaging the need for the provision of post-trial care. Nevertheless, all 
participants will be provided with an emergency contact number to reach the study 

IRAS ID: 316275 V.2 04/1/2022 
18 
 
 
 
investigators so that they can receive the necessary support when they have any questions 
or problems. 
 
Patient and Public Involvement (PPI) 
The research protocol was developed during the COVID Omicron wave, February-March 
2022, thus it was impossible to reach the patients and members of the public and get their 
input into the protocol. 
 
Discussion 
Following the extraction of teeth their supporting alveolar bone undergoes remodelling, which 
can result in the loss of height and width of the bony ridge; this is most marked in the aesthetic 
zone (anterior aspect of the upper jaw, the maxilla) (34,35). 
To minimise this loss of ridge volume, the ridge preservation technique has been developed, 
which involves the insertion of bone substitute material to act as a space maintainer and 
scaffold for new bone growth (36–38) 
The commonly used materials include bovine bone, human-donated bone and synthetic 
materials (6,38). There is a growing demand for non-animal products and the current synthetic 
materials have a high substitution rate which defeats the purpose of maintaining the space 
over periods longer than 4 to 5 months(10,12) . Bioactive glass (BAG) is a synthetic material 
with a low substitution rate, and antibacterial properties and is widely used by orthopaedic and 
ENT surgeons, but not yet by dental implant surgeons (13,14). This study aims to investigate 
whether the BAG material can be as effective or more effective as the bovine bone material 
as a bone substitute in implant dentistry. The importance of this study lies in its potential to 
facilitate a good ridge preservation and prevent more complicated augmentation procedures 
due to the loss of ridge volume in cases such as those listed below: 
1. Aesthetic zone when immediate or early (4-8 weeks) placement is not possible (39) 
2. Posterior maxilla – roots close to the floor of the sinus (40) 
3. Posterior mandible – roots close to inferior dental canal/ mental foramen 
4. Pontic area for aesthetics of the bridge 
5. Extraction of the tooth adjacent to the implant 
6. Large socket e.g., a molar with septal bone loss leaving a wide defect 

IRAS ID: 316275 V.2 04/1/2022 
19 
 
 
 
In addition to the ridge preservation, benefits of BAG include reducing the risk of more 
complex, expensive, and risky augmentation procedures. The use of BAG can benefit patients 
undergoing sinus augmentation procedures, as well as the treatment of peri-implantitis 
(infection and bone loss around dental implants) due to its osteoconductive, osteostimulative, 
and antibacterial properties. . 
 
Conclusion 
Ridge preservation is a well-documented technique for maintaining the bone volume of the 
alveolar ridges of the jaws. This study will assess the effectiveness of the use of the synthetic 
material BAG which presents no cross-infection risk and has documented antibacterial 
properties and the desired low substitution rate. The results of this pilot study will  provide the 
essential information to design a full-scale RCT and apply for funding too. Furthermore, the 
results of the pilot could be considered as the basis of further research into BAG use for general 
augmentation and in the treatment of peri-implantitis. 
 
Protocol version 
 2.0 
 
Funding 
Not funded 
 
Acknowledgements 
 
In preparation of this protocol, Dr Miland Joshi acted as a critical friend in the development of 
the statistical analysis plan, so the research team would like to acknowledge this support. The 
research team is thankful to Dr Izabella Penier for editing the manuscript. 
 
 
Author contributions 
 
Conceptualisation: Fadi N Barrak, Andrea Manfrin 
 
Co-authors: Aderonke, Ajiboye, Julian R Jones, Seiwi Li. 
Data curation: Andrea Manfrin 
Formal analysis: Andrea Manfrin 
Funding acquisition: None 

IRAS ID: 316275 V.2 04/1/2022 
20 
 
 
 
Methodology: Fadi N Barrak, Aderonke, Ajiboye, Andrea Manfrin 
 
Project administration: Aderonke, Ajiboye 
 
Visualisation: Fadi N Barrak, Andrea Manfrin 
 
Writing – original draft: Fadi N Barrak, Aderonke, Ajiboye, Andrea Manfrin 
 
Writing – review & editing: Fadi N Barrak, Aderonke, Ajiboye, R Jones, Seiwi Li, Andrea 
Manfrin 
 
References 
 
1. 
Elani HW, Starr JR, Da Silva JD, Gallucci GO. Trends in dental implant use in the US, 
1999–2016, and projections to 2026. Journal of dental research. 2018 Dec;97(13):1424- 
30. Available from: https://doi.org/10.1177/0022034518792567 
 
2. 
Cha HS, Kim JW, Hwang JH, Ahn KM. Frequency of bone graft in implant surgery. 
Maxillofacial plastic and reconstructive surgery. 2016 Dec;38(1):1-4. Available from: 
https://doi.org/10.1177/2041731418776819 
3. 
Caracaş RE, Manolea HO, Mitruţ I, Caracaş AM, Sălan AI, Drăghici MA, Rîcă AM. 
Frequency of Bone Augmentation Materials Use in a General Dental Practice. Current 
Health 
Sciences 
Journal. 
2021 
Jul;47(3):438. 
Available 
from: 
https://dx.doi.org/10.12865%2FCHSJ.47.03.16 
4. 
Yamada M, Egusa H. Current bone substitutes for implant dentistry. Journal of 
prosthodontic 
research. 
2018;62(2):152-61. 
Available 
from: 
https://doi.org/10.1016/j.jpor.2017.08.010 
5. 
Starch-Jensen T, Mordenfeld A, Becktor JP, Jensen SS. Maxillary sinus floor 
augmentation with synthetic bone substitutes compared with other grafting materials: A 
systematic review and meta-analysis. Implant Dentistry. 2018 Jun 1;27(3):363-74. 
Available from: https://doi.org/10.1097/id.0000000000000768 
6. 
Zhao R, Yang R, Cooper PR, Khurshid Z, Shavandi A, Ratnayake J. Bone grafts and 
substitutes in dentistry: A review of current trends and developments. Molecules. 2021 
Jan;26(10):3007. Available from: https://doi.org/10.3390/molecules26103007 

IRAS ID: 316275 V.2 04/1/2022 
21 
 
 
 
7. 
Darby I, Chen S, De Poi R. Ridge preservation: what is it and when should it be 
considered. Australian dental journal. 2008 Mar;53(1):11-21. 
8. Kalsi AS, Kalsi JS, Bassi S. Alveolar ridge preservation: why, when and how. British Dental 
Journal. 2019 Aug;227(4):264-74. Available from: https://doi.org/10.1038/s41415-019- 
0647-2 
9. 
Özkan Y, Akoglu B, Kulak-Özkan Y. Maxillary sinus floor augmentation using bovine bone 
grafts with simultaneous implant placement: a 5-year prospective follow-up study. Implant 
Dentistry. 
2011 
Dec 
1;20(6):455-9. 
Available 
from: 
https://doi.org/10.1097/id.0b013e3182386cbc 
10. Fernández RF, Bucchi C, Navarro P, Beltrán V, Borie E. Bone grafts utilized in dentistry: 
an analysis of patients' preferences. BMC Medical Ethics. 2015 Dec;16(1):1-6. Available 
from: https://doi.org/10.1186/s12910-015-0044-6 
11. Almutairi AS. A descriptive analysis of patient’s preferences in bone graft therapy in 
dentistry. International Journal of Health Sciences. 2019 May;13(3):24. 
12. Fernandez de Grado G, Keller L, Idoux-Gillet Y, Wagner Q, Musset AM, Benkirane-Jessel 
N, Bornert F, Offner D. Bone substitutes: a review of their characteristics, clinical use, and 
perspectives for large bone defects management. Journal of tissue engineering. 2018 Jun 
2;9:2041731418776819.Available from: https://doi.org/10.1177/2041731418776819 
13. Lindfors NC, Hyvönen P, Nyyssönen M, Kirjavainen M, Kankare J, Gullichsen E, Salo J. 
Bioactive glass S53P4 as bone graft substitute in treatment of osteomyelitis. Bone. 2010 
Aug 1;47(2):212-8. Available from: https://doi.org/10.1016/j.bone.2010.05.030 
14. Cannio M, Bellucci D, Roether JA, Boccaccini D, Cannillo V. Bioactive Glass Applications: 
A Literature Review of Human Clinical Trials. Materials. 2021 Jan;14(18):5440. Available 
from: https://doi.org/10.3390/ma14185440 
15. Mistry S, Kundu D, Datta S, Basu D. Comparison of bioactive glass coated and 
hydroxyapatite coated titanium dental implants in the human jaw bone. Australian dental 
journal. 
2011 
Mar;56(1):68-75. 
Available 
from: 
https://doi.org/10.1111/j.1834- 
7819.2010.01305.x 
16. Peltola M, Kinnunen I, Aitasalo K. Reconstruction of orbital wall defects with bioactive 
glass plates. Journal of Oral and Maxillofacial Surgery. 2008 Apr 1;66(4):639-46 Available 
from: https://doi.org/10.1016/j.joms.2007.11.019 

IRAS ID: 316275 V.2 04/1/2022 
22 
 
 
 
17. ASA Physical Status Classification System | American Society of Anaesthesiologists 
(ASA) [Internet]. Available from: https://www.asahq.org/standards-and-guidelines/asa- 
physical-status-classification-system 
18. Bonalive granules - S53P4 bioactive glass - bone regeneration [Internet]. Available from: 
https://www.bonalive.com/en/products/bonalive-granules/ 
19. Baino F, Hamzehlou S, Kargozar S. Bioactive glasses: where are we and where are we 
going? Journal of functional biomaterials. 2018 Mar;9(1):25. Available from: 
www.mdpi.com/journal/jfb 
20. Geistlich UK - User Benefits [Internet]. [cited 2022 May 16]. Available from: 
https://www.geistlich.co.uk/en/dental/bone-substitutes/bio-oss/user-benefits/ 
21. Orsini G, Traini T, Scarano A, Degidi M, Perrotti V, Piccirilli M, Piattelli A. Maxillary sinus 
augmentation with Bio‐Oss® particles: A light, scanning, and transmission electron 
microscopy study in man. Journal of Biomedical Materials Research Part B: Applied 
Biomaterials: An Official Journal of The Society for Biomaterials, The Japanese Society 
for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for 
Biomaterials. 2005 Jul;74(1):448-57. Available from: https://doi.org/10.1002/jbm.b.30196 
22. Kim YJ, Saiki CE, Silva K, Massuda CK, de Souza Faloni AP, Braz-Silva PH, Pallos D, 
Sendyk WR. Bone formation in grafts with Bio-Oss and autogenous bone at different 
proportions in rabbit calvaria. International Journal of Dentistry. 2020 Feb 19;2020. 
Available from: https://doi.org/10.1155/2020/2494128 
23. Caplanis N, Lozada JL, Kan JY. Extraction defect assessment, classification, and 
management. Cda Journal. 2005 Nov;33(11). 
24. Shi X, Nommeots-Nomm A, Todd NM, Devlin-Mullin A, Geng H, Lee PD, Mitchell CA, 
Jones JR. Bioactive glass scaffold architectures regulate patterning of bone regeneration 
in vivo. Applied Materials Today. 2020 Sep 1; 20:100770. Available from: 
https://doi.org/10.1016/j.apmt.2020.100770 
25. Li S, Tallia F, Mohammed AA, Stevens MM, Jones JR. Scaffold channel size influences 
stem cell differentiation pathway in 3-D printed silica hybrid scaffolds for cartilage 
regeneration. 
Biomaterials 
science. 
2020;8(16):4458-66. 
Available 
from: 
https://doi.org/10.1039/C9BM01829H 

IRAS ID: 316275 V.2 04/1/2022 
23 
 
 
 
26.  Solakoglu Ö, Götz W, Heydecke G, Schwarzenbach H. Histological and 
immunohistochemical comparison of two different allogeneic bone grafting materials for 
alveolar ridge reconstruction: a prospective randomized trial in humans. Clinical implant 
dentistry 
and 
related 
research. 
2019 
Oct;21(5):1002-16. 
Available 
from: 
https://doi.org/10.1111/cid.12824 
27. Charles P, Giraudeau B, Dechartres A, Baron G, Ravaud P. Reporting of sample size 
calculation 
in 
randomised 
controlled 
trials. 
Bmj. 
2009 
May 
12;338. 
https://doi.org/10.1136/bmj.b1732 
28. Rignon-Bret C, Hadida A, Aidan A, Nguyen TH, Pasquet G, Fron-Chabouis H, Wulfman 
C. Efficacy of bone substitute material in preserving volume when placing a maxillary 
immediate complete denture: study protocol for the PANORAMIX randomized controlled 
trial. Trials. 2016 Dec;17(1):1-2. Available from: https://doi.org/10.1186/s13063-016- 
1380-7  
29. Thabane L, Ma J, Chu R, Cheng J, Ismaila A, Rios LP, Robson R, Thabane M, 
Giangregorio L, Goldsmith CH. A tutorial on pilot studies: the what, why and how. BMC 
medical research methodology. 2010 Dec; 10:1-0. https://doi.org/10.1186/1471-2288-10-
1 
30. Hertzog MA. Considerations in determining sample size for pilot studies. Research in 
nursing & health. 2008 Apr;31(2):180-91. DOI: 10.1002/nur.20247 
31. Whitehead AL, Julious SA, Cooper CL, Campbell MJ. Estimating the sample size for a 
pilot randomised trial to minimise the overall trial sample size for the external pilot and 
main trial for a continuous outcome variable. Statistical methods in medical research. 
2016 Jun;25(3):1057-73. https://doi.org/10.1177/0962280215588241 
32. Simple randomisation service | Sealed Envelope [Internet]. [cited 2023 May 10]. Available 
from: https://www.sealedenvelope.com/simple-randomiser/v1/  
33. Zhang S, Paul J, Nantha-Aree M, Buckley N, Shahzad U, Cheng J, DeBeer J, Winemaker 
M, Wismer D, Punthakee D, Avram V. Empirical comparison of four baseline covariate 
adjustment methods in analysis of continuous outcomes in randomized controlled trials. 
Clinical epidemiology. 2014 Jul 14:227-35. https://doi.org/10.2147/clep.s56554  
34. Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and soft tissue contour 
changes following single-tooth extraction: a clinical and radiographic 12-month 
prospective study. International Journal of Periodontics & Restorative Dentistry. 2003 Aug 

IRAS ID: 316275 V.2 04/1/2022 
24 
 
 
1;23(4). 
35. Araújo, M.G. and Lindhe, J., 2005. Dimensional ridge alterations following tooth 
extraction. An experimental study in the dog. Journal of clinical periodontology, 32(2), 
pp.212-218.Available from: https://doi.org/10.1111/j.1600-051x.2005.00642.x 
36. Atieh MA, Alsabeeha NH, Payne AG, Ali S, Clovis Jr M, Esposito M. Interventions for 
replacing missing teeth: alveolar ridge preservation techniques for dental implant site 
development. Cochrane Database of Systematic Reviews. 2021(4). Available from: 
https://www.cochranelibrary.com/cdsr/doi/10.1002/14651858.CD010176.pub2/full 
37. Lee J, Yun J, Kim JJ, Koo KT, Seol YJ, Lee YM. Retrospective study of alveolar ridge 
preservation compared with no alveolar ridge preservation in periodontally compromised 
extraction sockets. International journal of implant dentistry. 2021 Dec;7(1):1-0. Available 
from: https://doi.org/10.1186/s40729-021-00305-2 
38. Kumar P, Vinitha B, Fathima G. Bone grafts in dentistry. Journal of pharmacy & bioallied 
sciences. 2013 Jun;5(Suppl 1): S125. Available from: https://doi.org/10.4103/0975- 
7406.113312 

IRAS ID: 316275 V.2 04/1/2022 
25 
 
 
 
39. Jung RE, Ioannidis A, Hämmerle CH, Thoma DS. Alveolar ridge preservation in the 
esthetic zone. 
Periodontology 
2000. 
2018 
Jun;77(1):165-75. 
Available 
from: 
https://doi.org/10.1111/prd.12209 
40. Cha JK, Song YW, Park SH, Jung RE, Jung UW, Thoma DS. Alveolar ridge preservation 
in the posterior maxilla reduces vertical dimensional change: a randomized controlled 
clinical trial. Clinical Oral Implants Research. 2019 Jun;30(6):515-23. Available from: 
https://doi.org/10.1111/clr.13436 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

IRAS ID: 316275 V.2 04/1/2022 
26 
 
 
Appendix 
 
Appendix 1 ASA physical status classification system 

IRAS ID: 316275 V.2 04/1/2022 
27 
 
 
 
 
 
American Society of Anaesthesiologists (2022) ASA Physical Status Classification 

IRAS ID: 316275 V.2 04/1/2022 
28 
 
 
 
Appendix 2 Instructions for the use of Bonalive 
 
 
 
 
Appendix 3 Instructions for the use of Bio-Oss® 
 
• 
Cut the Geistlich Bio-Oss® Collagen block to the appropriate size. 
• 
Apply it dry or moisten with saline solution. 
• 
When moistened, mould to the desired shape. 
• 
Place in defect site with forceps. 

IRAS ID: 316275 V.2 04/1/2022 
29 
 
 
 
• 
Avoid excessive compression. 
 
 
Appendix 4 Packaging instruction 
 
 
P650 
PACKING INSTRUCTION 
P650 
This packing instruction applies to UN No. 3373. 
1. 
The packaging shall be of good quality, strong enough to withstand the
shocks and loadings normally encountered during carriage, including transhipment
between cargo transport units and between cargo transport units and warehouses as
well as any removal from a pallet or overpack for subsequent manual or mechanical
handling. Packagings shall be constructed and closed to prevent any loss of contents
that might be caused under normal conditions of carriage by vibration or by changes
in temperature, humidity, or pressure. 
2. 
The packaging shall consist of at least three components: 
0. 
a primary receptacle. 
1. 
a secondary packaging; and 
2. 
an outer packaging 
3. 
Primary receptacles shall be packed in secondary packagings in such a way
that, under normal conditions of carriage, they cannot break, be punctured or leak
their contents into the secondary packaging. Secondary packagings shall be secured
in outer packagings with suitable cushioning material. Any leakage of the contents
shall not compromise the integrity of the cushioning material or of the outer
packaging. 
4. 
For carriage, the mark illustrated below shall be displayed on the external
surface of the outer packaging on a background of a contrasting colour and shall be
clearly visible and legible. The mark shall be in the form of a square set at an angle
of 45° (diamond-shaped) with minimum dimensions of 50 mm by 50 mm; the width
of the line shall be at least 2 mm and the letters and numbers shall be at least 6 mm
high. The proper shipping name "BIOLOGICAL SUBSTANCE, CATEGORY B" in
letters at least 6 mm high shall be marked on the outer packaging adjacent to the
diamond-shaped mark. 
5. 
of which either the secondary or the outer packaging shall be rigid. 
(5) At least one surface of the outer packaging shall have a minimum dimension of 100 
mm  100 mm. 
he completed package shall be capable of successfully passing the drop test in 6.3.5.3 as 
specified in 6.3.5.2 at a height of 1.2 m. Following the appropriate drop sequence, there shall 
be no leakage from the primary receptacle(s) which shall remain protected by absorbent 
material, when required, in the secondary packaging. 

IRAS ID: 316275 V.2 04/1/2022 
30 
 
 
 
P650 
PACKING INSTRUCTION (cont'd) 
P650 
1. 
For liquid substances: 
0. 
The primary receptacle(s) shall be leakproof; 
1. 
The secondary packaging shall be leakproof; 
2. 
If multiple fragile primary receptacles are placed in a single secondary
packaging, they shall be either individually wrapped or separated to prevent
contact between them; 
3. 
Absorbent material shall be placed between the primary receptacle(s)
and the secondary packaging. The absorbent material shall be in quantity
sufficient to absorb the entire contents of the primary receptacle(s) so that
any release of the liquid substance will not compromise the integrity of the
cushioning material or of the outer packaging; 
4. 
The primary receptacle or the secondary packaging shall be capable
of withstanding, without leakage, an internal pressure of 95 kPa (0.95 bar). 
1. 
For solid substances: 
0. 
The primary receptacle(s) shall be siftproof; 
1. 
The secondary packaging shall be siftproof; 
2. 
If multiple fragile primary receptacles are placed in a single secondary
packaging, they shall be either individually wrapped or separated to prevent
contact between them; 
3. 
If there is any doubt as to whether or not residual liquid may be present
in the primary receptacle during carriage then a packaging suitable for liquids,
including absorbent materials, shall be used. 
1. 
Refrigerated or frozen specimens: Ice, dry ice and liquid nitrogen: 
0. 
When dry ice or liquid nitrogen is used as a coolant, the requirements
of 5.5.3 shall apply. When used, ice shall be placed outside the secondary
packagings or in the outer packaging or an overpack. Interior supports shall
be provided to secure the secondary packagings in the original position. If ice
is used, the outside packaging or overpack shall be leakproof. 
1. 
The primary receptacle and the secondary packaging shall maintain 
their integrity at the temperature of the refrigerant used as well as the
temperatures and the pressures which could result if refrigeration were lost. 
(10) When packages are placed in an overpack, the package marks required by this 
packing instruction shall either be clearly visible or be reproduced on the outside of the 
overpack. 
(11) Infectious substances assigned to UN No. 3373 which are packed and packages 
which are marked in accordance with this packing instruction are not subject to any other 
requirement in ADR. 
(12) Clear instructions on filling and closing such packages shall be provided by
packaging manufacturers and subsequent distributors to the consignor or to the person who
prepares the package (e.g. patient) to enable the package to be correctly prepared for 
carriage. 
(13) Other dangerous goods shall not be packed in the same packaging as Class 6.2
infectious substances unless they are necessary for maintaining the viability, stabilising or
preventing degradation or neutralising the hazards of the infectious substances. A quantity
of 30 ml or less of dangerous goods included in Classes 3, 8 or 9 may be packed in each
primary receptacle containing infectious substances. When these small quantities of 
dangerous goods are packed with infectious substances in accordance with this packing
instruction no other requirements of ADR need be met. 

IRAS ID: 316275 V.2 04/1/2022 
31 
 
 
 
 
 
Appendix 5 Biological substance category label 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Appendix 6: HTA License for ICHTB 
(14) If any substance has leaked and has been spilled in a cargo transport unit, it may 
not be reused until after it has been thoroughly cleaned and, if necessary, disinfected or
decontaminated. Any other goods and articles carried in the same vehicle or container shall
be examined for possible contamination. 

IRAS ID: 316275 V.2 04/1/2022 
32 
 
 
 
 
 
 
Appendix 7 Dry ice label 

IRAS ID: 316275 V.2 04/1/2022 
33 
 
 
 
 

IRAS ID: 316275 V.2 04/1/2022 
34 
 
 
 
Appendix 8 Email from MHRA 
 

IRAS ID: 316275 V.2 04/1/2022 
35